One problem, multiple potential targets: Where are we now in the development of small molecule inhibitors against Shiga toxin?

Shiga toxin-producing Escherichia coli (STEC) are a group of enteric pathogens which carry phage-encoded Shiga toxins (Stx). STEC infections begin with severe abdominal pain and non-bloody diarrhoea, which can progress to bloody diarrhoea after approximately 4-days post-infection. In high-risk groups such as children and the elderly, patients may develop haemolytic uremic syndrome (HUS). HUS is characterised by microangiopathic haemolytic anaemia, thrombocytopenia, and in severe disease acute renal failure. Traditional antibiotics have been linked with increased toxin production due to the activation of recA-mediated bacterial stress response, resulting in poorer patient outcomes. Therefore, treatment relies on supportive therapies. Antivirulence strategies have been explored as an alternative treatment for bacterial infections and blockers of virulence factors such as the Type III Secretion System. Recent improvements in the mechanistic understanding of the Stx pathway have led to the design of inhibitors to disrupt the pathway, leading to toxin-mediated ribosome damage. However, compounds have yet to progress beyond Phase III clinical trials successfully. This review explores the progress in developing small molecule inhibitors by collating lead compounds derived from in-silico and experimental approaches.

Fragment Screening to Identify Inhibitors Targeting Ribosome Binding of Shiga Toxin 2.

Shiga toxins are the main virulence factors of Shiga toxin producing E. coli (STEC) and S. dysenteriae. There is no effective therapy to counter the disease caused by these toxins. The A1 subunits of Shiga toxins bind the C-termini of ribosomal P-stalk proteins to depurinate the sarcin/ricin loop. The ribosome binding site of Shiga toxin 2 has not been targeted by small molecules. We screened a fragment library against the A1 subunit of Shiga toxin 2 (Stx2A1) and identified a fragment, BTB13086, which bound at the ribosome binding site and mimicked the binding mode of the P-stalk proteins. We synthesized analogs of BTB13086 and identified a series of molecules with similar affinity and inhibitory activity. These are the first compounds that bind at the ribosome binding site of Stx2A1 and inhibit activity. These compounds hold great promise for further inhibitor development against STEC infection.

RNA-Seq analysis of long non-coding RNA in human intestinal epithelial cells infected by Shiga toxin-producing Escherichia coli.

BACKGROUND: The Shiga toxin-producing Escherichia coli (STEC) infects animals and induces acute intestinal inflammation. Long non-coding RNAs (lncRNAs) are known to play crucial roles in modulating inflammation response. However, it is not clear whether lncRNAs are involved in STEC-induced inflammation. METHODS AND RESULTS: To understand the association of lncRNAs with STEC infection, we used RNA-seq technology to analyze the profiles of lncRNAs in Mock-infected and STEC-infected human intestinal epithelial cells (HIECs). We detected a total of 702 lncRNAs differentially expressed by STEC infection. 583 differentially expressed lncRNAs acted as competitive microRNAs (miRNAs) binding elements in regulating the gene expression involved in TNF signaling pathway, IL-17 signaling pathway, PI3K-Akt signaling pathway, and apoptosis pathways. We analyzed 3 targeted genes, TRADD, TRAF1 and TGFB2, which were differentially regulated by mRNA-miRNA-lncRNA interaction network, potentially involved in the inflammatory and apoptotic response to STEC infection. Functional analysis of up/downstream genes associated with differentially expressed lncRNAs revealed their role in adheres junction and endocytosis. We also used the qRT-PCR technique to validate 8 randomly selected differentially expressed lncRNAs and mRNAs in STEC-infected HIECs. CONCLUSION: Our results, for the first time, revealed differentially expressed lncRNAs induced by STEC infection of HIECs. The results will help investigate the molecular mechanisms for the inflammatory responses induced by STEC.

Gb3-Coated Bovine Milk Exosomes as a Practical Neutralizer for Shiga Toxin.

Shiga toxin (Stx) is associated with foodborne infections of some Shigella spp. and Shiga toxin-producing Escherichia coli (STEC), leading to life-threatening hemolytic uremic syndrome (HUS). Target-specific therapeutics against HUS are currently unavailable in clinical practice. Herein, we reported the construction and in vitro characterization of Gb3-coated bovine milk exosomes (Gb3-mExo) as a multivalent Shiga toxin neutralizer, utilizing the natural advantages of milk exosomes (mExo) in drug delivery and multivalent interactions between Stx and its receptor Gb3. Gb3-mExo constructs were achieved by conjugating mExo with the Gb3 derivatives containing stearic acid-derived lipid tail, which was prepared through an efficient chemoenzymatic approach. The constructs were able to potently neutralize the binding of the B subunit of Stx2 (Stx2B) to receptor Gb3 immobilized on the plate or expressed on model cells. General safety of the constructs was evidenced by the cytotoxicity analysis and hemolysis assay. In addition to the excellent stability under conventional storage and handling conditions, the construct can also retain most of its neutralization potency under gastrointestinal pH extremes, showing the potential for oral administration. Considering the natural availability and excellent biocompatibility of mExo, Gb3-mExo conjugates should prove to be a practical prophylactic and therapeutic for the Shiga toxin-related infections.

Shiga toxin targets the podocyte causing hemolytic uremic syndrome through endothelial complement activation.

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli hemolytic uremic syndrome (STEC-HUS) is the leading cause of acute kidney injury in children, with an associated mortality of up to 5%. The mechanisms underlying STEC-HUS and why the glomerular microvasculature is so susceptible to injury following systemic Stx infection are unclear. METHODS: Transgenic mice were engineered to express the Stx receptor (Gb3) exclusively in their kidney podocytes (Pod-Gb3) and challenged with systemic Stx. Human glomerular cell models and kidney biopsies from patients with STEC-HUS were also studied. FINDINGS: Stx-challenged Pod-Gb3 mice developed STEC-HUS. This was mediated by a reduction in podocyte vascular endothelial growth factor A (VEGF-A), which led to loss of glomerular endothelial cell (GEnC) glycocalyx, a reduction in GEnC inhibitory complement factor H binding, and local activation of the complement pathway. Early therapeutic inhibition of the terminal complement pathway with a C5 inhibitor rescued this podocyte-driven, Stx-induced HUS phenotype. CONCLUSIONS: This study potentially explains why systemic Stx exposure targets the glomerulus and supports the early use of terminal complement pathway inhibition in this devastating disease. FUNDING: This work was supported by the UK Medical Research Council (MRC) (grant nos. G0901987 and MR/K010492/1) and Kidney Research UK (grant nos. TF_007_20151127, RP42/2012, and SP/FSGS1/2013). The Mary Lyon Center is part of the MRC Harwell Institute and is funded by the MRC (A410).

Expression of hes, iha, and tpsA codified in locus of adhesion and autoaggregation and their involvement in the capability of shiga toxin-producing Escherichia coli strains to adhere to epithelial cells.

OBJECTIVES: Shiga toxin-producing Escherichia coli strains LAA-positive are important cause of human infection. The capability to adhere to epithelial cells is a key virulence trait, and genes codified in LAA pathogenicity island could be involved in the adhesion during the pathogenesis of LAA-positive STEC strains. Thus, our objectives were to compare hes-negative and hes-positive STEC strains in their adherence capability to epithelial cells (HEp-2) and to evaluate the expression levels of the hes, iha, and tpsA in the bacteria adhered and non-adhered to HEp-2 cells. These genes are encoded in LAA, and are virulence factors that participate in adhesion and autoaggregation. RESULTS: We could not observe differences between the adhesion of strains but also in the expression level of of hes, iha, and tpsA. Genes encoded in LAA contribute to the adhesion phenotype though the expression of STEC adhesins is a coordinated event that depends not only the strain but also on the environment as well as its genetic background. Therefore, the results of this study suggest that LAA ,the most prevalent PAI among LEE-negative STEC strains, plays a role in pathogenesis.

Hyperhydration to Improve Kidney Outcomes in Children with Shiga Toxin-Producing E. coli Infection: a multinational embedded cluster crossover randomized trial (the HIKO STEC trial).

BACKGROUND: Shiga toxin-producing E. coli (STEC) infections affect children and adults worldwide, and treatment remain solely supportive. Up to 15-20% of children infected by high-risk STEC (i.e., E. coli that produce Shiga toxin 2) develop hemolytic anemia, thrombocytopenia, and kidney failure (i.e., hemolytic uremic syndrome (HUS)), over half of whom require acute dialysis and 3% die. Although no therapy is widely accepted as being able to prevent the development of HUS and its complications, several observational studies suggest that intravascular volume expansion (hyperhydration) may prevent end organ damage. A randomized trial is needed to confirm or refute this hypothesis. METHODS: We will conduct a pragmatic, embedded, cluster-randomized, crossover trial in 26 pediatric institutions to determine if hyperhydration, compared to conservative fluid management, improves outcomes in 1040 children with high-risk STEC infections. The primary outcome is major adverse kidney events within 30 days (MAKE30), a composite measure that includes death, initiation of new renal replacement therapy, or persistent kidney dysfunction. Secondary outcomes include life-threatening, extrarenal complications, and development of HUS. Pathway eligible children will be treated per institutional allocation to each pathway. In the hyperhydration pathway, all eligible children are hospitalized and administered 200% maintenance balanced crystalloid fluids up to targets of 10% weight gain and 20% reduction in hematocrit. Sites in the conservative fluid management pathway manage children as in- or outpatients, based on clinician preference, with the pathway focused on close laboratory monitoring, and maintenance of euvolemia. Based on historical data, we estimate that 10% of children in our conservative fluid management pathway will experience the primary outcome. With 26 clusters enrolling a mean of 40 patients each with an intraclass correlation coefficient of 0.11, we will have 90% power to detect a 5% absolute risk reduction. DISCUSSION: HUS is a devastating illness with no treatment options. This pragmatic study will determine if hyperhydration can reduce morbidity associated with HUS in children with high-risk STEC infection. TRIAL REGISTRATION: ClinicalTrials.gov NCT05219110 . Registered on February 1, 2022.

Shiga Toxin (Stx) Type 1a and Stx2a Translocate through a Three-Layer Intestinal Model.

Shiga toxins (Stxs) produced by ingested E. coli can induce hemolytic uremic syndrome after crossing the intact intestinal barrier, entering the bloodstream, and targeting endothelial cells in the kidney. The method(s) by which the toxins reach the bloodstream are not fully defined. Here, we used two polarized cell models to evaluate Stx translocation: (i) a single-layer primary colonic epithelial cell model and (ii) a three-cell-layer model with colonic epithelial cells, myofibroblasts, and colonic endothelial cells. We traced the movement of Stx types 1a and 2a across the barrier models by measuring the toxicity of apical and basolateral media on Vero cells. We found that Stx1a and Stx2a crossed both models in either direction. However, approximately 10-fold more Stx translocated in the three-layer model as compared to the single-layer model. Overall, the percentage of toxin that translocated was about 0.01% in the epithelial-cell-only model but up to 0.09% in the three-cell-layer model. In both models, approximately 3- to 4-fold more Stx2a translocated than Stx1a. Infection of the three-cell-layer model with Stx-producing Escherichia coli (STEC) strains showed that serotype O157:H7 STEC reduced barrier function in the model and that the damage was not dependent on the presence of the eae gene. Infection of the three-layer model with O26:H11 STEC strain TW08571 (Stx1a+ and Stx2a+), however, allowed translocation of modest amounts of Stx without reducing barrier function. Deletion of stx2a from TW08571 or the use of anti-Stx1 antibody prevented translocation of toxin. Our results suggest that single-cell models may underestimate the amount of Stx translocation and that the more biomimetic three-layer model is suited for Stx translocation inhibitor studies.

Optimized Bioorthogonal Non-canonical Amino Acid Tagging to Identify Serotype-Specific Biomarkers in Verotoxigenic Escherichia coli.

According to Canada's Food Report Card 2016, there are 4 million foodborne illnesses acquired each year in the nation alone. The leading causes of foodborne illness are pathogenic bacteria such as shigatoxigenic/verotoxigenic Escherichia coli (STEC/VTEC) and Listeria monocytogenes. Most current detection methods used to identify these bacterial pathogens are limited in their validity since they are not specific to detecting metabolically active organisms, potentially generating false-positive results from non-living or non-viable bacteria. Previously, our lab developed an optimized bioorthogonal non-canonical amino acid tagging (BONCAT) method which allows for the labeling of translationally active wild-type pathogenic bacteria. Incorporation of homopropargyl glycine (HPG) into the cellular surfaces of bacteria allows for protein tagging using the bioorthogonal alkyne handle to report on the presence of pathogenic bacteria. Here, we use proteomics to identify more than 400 proteins differentially detected by BONCAT between at least two of five different VTEC serotypes. These findings pave the way for future examination of these proteins as biomarkers in BONCAT-utilizing assays.

Nitrate enrichment does not affect enteropathogenic Escherichia coli in aquatic microcosms but may affect other strains present in aquatic habitats.

Eutrophication of the planet's aquatic systems is increasing at an unprecedented rate. In freshwater systems, nitrate-one of the nutrients responsible for eutrophication-is linked to biodiversity losses and ecosystem degradation. One of the main sources of freshwater nitrate pollution in New Zealand is agriculture. New Zealand's pastoral farming system relies heavily on the application of chemical fertilisers. These fertilisers in combination with animal urine, also high in nitrogen, result in high rates of nitrogen leaching into adjacent aquatic systems. In addition to nitrogen, livestock waste commonly carries human and animal enteropathogenic bacteria, many of which can survive in freshwater environments. Two strains of enteropathogenic bacteria found in New Zealand cattle, are K99 and Shiga-toxin producing Escherichia coli (STEC). To better understand the effects of ambient nitrate concentrations in the water column on environmental enteropathogenic bacteria survival, a microcosm experiment with three nitrate-nitrogen concentrations (0, 1, and 3 mg NO3-N /L), two enteropathogenic bacterial strains (STEC O26-human, and K99-animal), and two water types (sterile and containing natural microbiota) was run. Both STEC O26 and K99 reached 500 CFU/10 ml in both water types at all three nitrate concentrations within 24 hours and remained at those levels for the full 91 days of the experiment. Although enteropathogenic strains showed no response to water column nitrate concentrations, the survival of background Escherichia coli, imported as part of the in-stream microbiota did, surviving longer in 1 and 3 mg NO3-N/Lconcentrations (P < 0.001). While further work is needed to fully understand how nitrate enrichment and in-stream microbiota may affect the viability of human and animal pathogens in freshwater systems, it is clear that these two New Zealand strains of STEC O26 and K99 can persist in river water for extended periods alongside some natural microbiota.

Transduction of stx2a mediated by phage (Φ11-3088) from Escherichia coli O104:H4 in vitro and in situ during sprouting of mung beans.

Escherichia coli O104:H4 strain 11-3088 encoding Stx2a is epidemiologically related to the foodborne outbreak associated with sprouts in Germany, 2011. Sprouting provides suitable conditions for bacterial growth and may lead to transduction of non-pathogenic strains of E. coli with Stx phages. Although transduction of E. coli by Stx phages in food has been documented, data on the phages from E. coli O104:H4 is limited. This study determined the host range of the bacteriophage Φ11-3088 from E. coli O104:H4 using E. coli O104:H4 ∆stx2::gfp::ampr and demonstrated phage transduction during sprouting. The Φ11-3088∆stx transduced 5/45 strains, including generic E. coli, pap-positive E. coli O103:H2, ETEC, and S. sonnei. The expression level of Φ11-3088∆stx differed among lysogens upon induction. Of the 3 highly induced lysogens, the lytic cycle was induced in E. coli O104:H4∆stx2::gfp::ampr and O103:H2 but not in S. sonnei. E. coli DH5α was the only strain susceptible to lytic infection by Φ11-3088∆stx. To explore the effect of drying and rehydration during seed storage and sprouting on phage induction and transduction, mung beans inoculated with the phage donor E. coli O104:H4∆stx2::gfp::ampr (8 log CFU/g) were dried, rehydrated, and incubated with the phage recipient E. coli DH5α (7 log CFU/g) for 96 h. Sprouted seeds harbored about 3 log CFU/g of putative lysogens that acquired ampicillin resistance. At the end of sprouting, 71 % of putative lysogens encoded gfp, confirming phage transduction. Overall, stx transfer by phages may increase the cell counts of STEC during sprouting by converting generic E. coli to STEC.

Development of a novel tetravalent peptide that absorbs subtilase cytotoxin by targeting the receptor-binding B-subunit.

Subtilase cytotoxin (SubAB) is a major virulence factor produced by eae-negative Shiga-toxigenic Escherichia coli (STEC) that can cause fatal systemic complications. SubAB binds to target cells through multivalent interactions between its B-subunit pentamer and receptor molecules such as glycoproteins with a terminal N-glycolylneuraminic acid (Neu5Gc). We screened randomized multivalent peptide libraries synthesized on a cellulose membrane and identified a series of tetravalent peptides that efficiently bind to the receptor-binding region of the SubAB B-subunit pentamer. These peptides competitively inhibited the binding of the B-subunit to a receptor-mimic molecule containing clustered Neu5Gc (Neu5Gc-polymer). We selected the peptide with the highest inhibitory efficacy, FFP-tet, and covalently bound it to beads to synthesize FFP-tet-beads, a highly clustered SubAB absorber that displayed potency to absorb SubAB cytotoxicity through direct binding to the toxin. The efficacy of FFP-tet-beads to absorb SubAB cytotoxicity in solution was similar to that of Neu5Gc-polymer, suggesting that FFP-tet-beads might be an effective therapeutic agent against complications arising from eae-negative STEC infection.

Detection of Shiga Toxin-Producing Escherichia coli (STEC) in the Endocervix of Asymptomatic Pregnant Women. Can STEC Be a Risk Factor for Adverse Pregnancy Outcomes?

The presence of Escherichia coli in the vaginal microbiome has been associated with pregnancy complications. In previous works, we demonstrated that Shiga toxin-producing Escherichia coli (STEC) can produce abortion and premature delivery in rats and that Shiga toxin type 2 (Stx2) can impair human trophoblast cell lines. The hypothesis of this work was that STEC may colonize the lower female reproductive tract and be responsible for adverse pregnancy outcomes. Thus, the aim of this work was to evaluate the presence and prevalence of virulence factor genes from STEC in the endocervix of asymptomatic pregnant women. For that purpose, endocervical swabs were collected from pregnant women during their prenatal examination. Swab samples were enriched in a differential medium to select Enterobacteria. Then, positive samples were analyzed by PCR to detect genes characteristic of Escherichia sp. (such as uidA and yaiO), genes specific for portions of the rfb (O-antigen-encoding) regions of STEC O157 (rfbO157), and STEC virulence factor genes (such as stx1, stx2, eae, lpfAO113, hcpA, iha, sab, subAB). The cytotoxic effects of stx2-positive supernatants from E. coli recovered from the endocervix were evaluated in Vero cells. Our results showed that 11.7% of the endocervical samples were positive for E. coli. Additionally, we found samples positive for stx2 and other virulence factors for STEC. The bacterial supernatant from an isolate identified as E. coli O113:NT, carrying the stx2 gene, exhibited cytotoxic activity in Vero, Swan 71 and Hela cells. Our results open a new perspective regarding the presence of STEC during pregnancy.

Characterization of a T4-like Bacteriophage vB_EcoM-Sa45lw as a Potential Biocontrol Agent for Shiga Toxin-Producing Escherichia coli O45 Contaminated on Mung Bean Seeds.

Application of lytic bacteriophages is a promising and alternative intervention technology to relieve antibiotic resistance pressure and control bacterial pathogens in the food industry. Despite the increase of produce-associated outbreaks caused by non-O157 Shiga toxin-producing E. coli (STEC) serogroups, the information of phage application on sprouts to mitigate these pathogens is lacking. Therefore, the objective of this study was to characterize a T4-like Escherichia phage vB_EcoM-Sa45lw (or Sa45lw) for the biocontrol potential of STEC O45 on mung bean seeds. Phage Sa45lw belongs to the Tequatrovirus genus under the Myoviridae family and displays a close evolutionary relationship with a STEC O157-infecting phage AR1. Sa45lw contains a long-tail fiber gene (gp37), sharing high genetic similarity with the counterpart of Escherichia phage KIT03, and a unique tail lysozyme (gp5) to distinguish its host range (STEC O157, O45, ATCC 13706, and Salmonella Montevideo and Thompson) from phage KIT03 (O157 and Salmonella enterica). No stx, antibiotic resistance, and lysogenic genes were found in the Sa45lw genome. The phage has a latent period of 27 min with an estimated burst size of 80 PFU/CFU and is stable at a wide range of pH (pH 3 to pH 10.5) and temperatures (-80°C to 50°C). Phage Sa45lw is particularly effective in reducing E. coli O45:H16 both in vitro (MOI = 10) by 5 log and upon application (MOI = 1,000) on the contaminated mung bean seeds for 15 min by 2 log at 25°C. These findings highlight the potential of phage application against non-O157 STEC on sprout seeds. IMPORTANCE Seeds contaminated with foodborne pathogens, such as Shiga toxin-producing E. coli, are the primary sources of contamination in produce and have contributed to numerous foodborne outbreaks. Antibiotic resistance has been a long-lasting issue that poses a threat to human health and the food industry. Therefore, developing novel antimicrobial interventions, such as bacteriophage application, is pivotal to combat these pathogens. This study characterized a lytic bacteriophage Sa45lw as an alternative antimicrobial agent to control pathogenic E. coli on the contaminated mung bean seeds. The phage exhibited antimicrobial effects against both pathogenic E. coli and Salmonella without containing virulent or lysogenic genes that could compromise the safety of phage application. In addition, after 15 min of phage treatment, Sa45lw mitigated E. coli O45:H16 on the contaminated mung bean seeds by a 2-log reduction at room temperature, demonstrating the biocontrol potential of non-O157 Shiga toxin-producing E. coli on sprout seeds.

Diagnosis and Treatment for Shiga Toxin-Producing Escherichia coli Associated Hemolytic Uremic Syndrome.

Shiga toxin-producing Escherichia coli (STEC)-associated hemolytic uremic syndrome (STEC-HUS) is a clinical syndrome involving hemolytic anemia (with fragmented red blood cells), low levels of platelets in the blood (thrombocytopenia), and acute kidney injury (AKI). It is the major infectious cause of AKI in children. In severe cases, neurological complications and even death may occur. Treating STEC-HUS is challenging, as patients often already have organ injuries when they seek medical treatment. Early diagnosis is of great significance for improving prognosis and reducing mortality and sequelae. In this review, we first briefly summarize the diagnostics for STEC-HUS, including history taking, clinical manifestations, fecal and serological detection methods for STEC, and complement activation monitoring. We also summarize preventive and therapeutic strategies for STEC-HUS, such as vaccines, volume expansion, renal replacement therapy (RRT), antibiotics, plasma exchange, antibodies and inhibitors that interfere with receptor binding, and the intracellular trafficking of the Shiga toxin.

Shiga toxin (Stx) type 2-induced increase in O-linked N-acetyl glucosamine protein modification: a new therapeutic target?

Shiga toxin (Stx)-producing Escherichia coli (STEC) causes bloody diarrhea, which may progress to the potentially fatal hemolytic uremic syndrome (HUS). Development of HUS after STEC infection is dependent on Stx, and is particularly linked to Stx type 2a, Stx2a (Melton-Celsa, 2014; Scheutz, 2014). In this issue of EMBO Molecular Medicine, Lee et al report that O-linked N-acetyl glucosamine protein modification (O-GlcNAcylation) is increased in host cells after Stx exposure and the subsequent endoplasmic reticulum (ER) stress response. The elevated O-GlcNAcylation resulted in elevated inflammatory and apoptotic processes. Inhibition of O-GlcNAcylation with OSMI-1 protected cells from the Stx2a-induced damage. In mice intoxicated with Stx2a, OSMI-1 treatment reduced kidney damage and increased mouse survival.

Delayed lactose utilization among Shiga toxin-producing Escherichia coli of serogroup O121.

Two outbreaks of Shiga toxin-producing Escherichia coli O121:H19 associated with wheat flour, in the United States of America and Canada, involved strains with an unusual phenotype, delayed lactose utilization (DLU). These strains do not ferment lactose when initially cultured on MacConkey agar (MAC), but lactose fermentation occurs following subculture to a second plate of MAC. The prevalence of DLU was determined by examining the ß-galactosidase activity of 49 strains of E. coli O121, and of 37 other strains of E. coli. Twenty four of forty three O121:H19 and one O121:NM displayed DLU. Two strains (O121:NM and O145:H34) did not have detectable ß-galactosidase activity. ß-glucuronidase activity of O121 strains was also determined. All but six DLU strains had normal ß-glucuronidase activity. ß-glucuronidase activity was suppressed on MAC for 17 of 23 O121 non-DLU strains. Genomic analysis found that DLU strains possessed an insertion sequence, IS600 (1267 bp), between lacZ (ß-galactosidase) and lacY (ß-galactoside permease), that was not present in strains exhibiting normal lactose utilization. The insert might reduce the expression of ß-galactoside permease, delaying import of lactose, resulting in the DLU phenotype. The high probability of DLU should be considered when using lactose-containing media for the isolation of STEC O121.

Psychoactive Drugs Induce the SOS Response and Shiga Toxin Production in Escherichia coli.

Several classes of non-antibiotic drugs, including psychoactive drugs, proton-pump inhibitors (PPIs), non-steroidal anti-inflammatory drugs (NSAIDs), and others, appear to have strong antimicrobial properties. We considered whether psychoactive drugs induce the SOS response in E. coli bacteria and, consequently, induce Shiga toxins in Shiga-toxigenic E. coli (STEC). We measured the induction of an SOS response using a recA-lacZ E. coli reporter strain, as RecA is an early, reliable, and quantifiable marker for activation of the SOS stress response pathway. We also measured the production and release of Shiga toxin 2 (Stx2) from a classic E. coli O157:H7 strain, derived from a food-borne outbreak due to spinach. Some, but not all, serotonin selective reuptake inhibitors (SSRIs) and antipsychotic drugs induced an SOS response. The use of SSRIs is widespread and increasing; thus, the use of these antidepressants could account for some cases of hemolytic-uremic syndrome due to STEC and is not attributable to antibiotic administration. SSRIs could have detrimental effects on the normal intestinal microbiome in humans. In addition, as SSRIs are resistant to environmental breakdown, they could have effects on microbial communities, including aquatic ecosystems, long after they have left the human body.

Shiga Toxins: An Update on Host Factors and Biomedical Applications.

Shiga toxins (Stxs) are classic bacterial toxins and major virulence factors of toxigenic Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC). These toxins recognize a glycosphingolipid globotriaosylceramide (Gb3/CD77) as their receptor and inhibit protein synthesis in cells by cleaving 28S ribosomal RNA. They are the major cause of life-threatening complications such as hemolytic uremic syndrome (HUS), associated with severe cases of EHEC infection, which is the leading cause of acute kidney injury in children. The threat of Stxs is exacerbated by the lack of toxin inhibitors and effective treatment for HUS. Here, we briefly summarize the Stx structure, subtypes, in vitro and in vivo models, Gb3 expression and HUS, and then introduce recent studies using CRISPR-Cas9-mediated genome-wide screens to identify the host cell factors required for Stx action. We also summarize the latest progress in utilizing and engineering Stx components for biomedical applications.

Isolation and Characterization of Shiga Toxin Bacteriophages.

Shiga toxin (Stx) phages can be induced from Stx-producing Escherichia coli strains (STEC) or can be isolated as free virions from different samples. Here we describe methods used for the detection, enumeration, and isolation of Stx bacteriophages. Stx phages are temperate phages located in the genome of STEC. Their induction from the host strain cultures is achieved by different inducing agents, mitomycin C being one of the most commonly used. Detection of infectious Stx phages requires the production of visible plaques in a confluent lawn of the host strain using a double agar layer method. However, as the plaques produced by Stx phages are often barely visible and there is a possibility that non-Stx phages can also be induced from the strain, a hybridization step should be added to recognize and properly enumerate the lysis plaques generated after induction. Molecular methods can also be used to identify and enumerate Stx phages. Real-time quantitative PCR (qPCR) is the most accurate method for absolute quantification, although it cannot determine the infectivity of Stx phages. qPCR can also be useful for the detection of free Stx phage virions in different sample types.Stx phages induced from lysogenic bacterial strains can be purified by cesium chloride density gradients; this protocol also helps to specifically discriminate Stx phages from other prophages present in the genome of the host strain by selecting the phages expressing the Stx gene. High titer suspensions of Stx phages obtained after induction of large volumes of bacterial cultures and lysate concentration permits phage characterization by electron microscopy studies and genomic analysis.