Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype

Shiga toxin-producing Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise.

Shiga toxin-producing Escherichia coli (STEC) are a group of bacterial organisms that are capable of producing one or more types of Shiga toxin (Stx). STEC are associated with a disease spectrum ranging from diarrhea and hemorrhagic colitis (HC) to the potentially fatal hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP). STEC infections are typically food-borne (Dupont, 2007) and the production of Shiga toxins (Stx1, Stx2 or a variant) is believed to be central to the pathogenesis of these organisms. STEC strains are the result of an insertion of one of a group of lysogenic lambdoid bacteriophages that harbor an Stx1/2-encoding gene into the E. coli genome. The clinical syndromes, pathogenic characteristics, the pathobiology of these organisms and the toxins they produce are reviewed in Melton-Celsa et al. (2012); Farrokh et al. (2013); Kruger and Lucchesi (2015).
In recent years, novel serotypes have emerged culminating in a major outbreak in 2011 caused by a novel pathotype, E. coli O104:H4. The review at hand focuses on potential treatment strategies for STEC infections in light of a consensus contraindication of employing antimicrobials for these bacterial pathogens. The rise of E. coli O104:H4 and approaches employed in its treatment are highlighted.

Emerging STEC Serotypes
A large number of STEC serotypes has been documented; these have been isolated from various types of animals including cattle, sheep, and goats (Farrokh et al., 2013). More than 380 STEC serotypes have been associated with human disease; some of the most frequently reported serotypes include O111:H-, O26:H11/H-, O103:H2, O113:H21, O91:H21/H-, O117:H7, O118:H16, O121:H19, O145:H28, O128:H2/H-, and O146:H21. The O157:H7 serotype has been the most commonly isolated one in association with HC and HUS in both outbreaks and sporadic cases. It accounts for more than 30% of estimated STEC illness and mortality cases in the United States (Karmali et al., 2010;Scallan et al., 2011). However, there are some indications that non-O157 STEC are gaining traction in the United States and that they may be even more common than O157 strains in severe illnesses caused by STEC in parts of Europe, Latin America, Australia, and Africa (Blanco et al., 2005;Wang et al., 2013).
The epidemiology and pathogenic characteristics of non-O157 serotypes are not well studied; however, the limited reported data indicates some differences between the two types of infections. Non-O157 strains appear to induce a longer period of diarrhea which is less frequently of the hemorrhagic type (Johnson et al., 2006). Nevertheless, studies demonstrate that these non-O157 serotypes can be as virulent as O157 serotypes depending on the strain involved (Ethelberg et al., 2004).
Perhaps highlighting the relevance of monitoring these non-O157 serotypes was the emergence of the rather notorious E. coli O104:H4. This novel pathogen was the cause of a 2011 outbreak that affected 16 European countries with the majority of cases reported in Germany. Few cases were reported in Canada and the United States as well; nevertheless, these were travelers who had been to Europe prior to becoming ill. Reports of this novel pathogen started in May of 2011 and had peaked and then dwindled by July of the same year due to control measures that were implemented. The WHO indicates that 4075 cases and 50 deaths were caused by this STEC outbreak. Therefore, a 1.23% mortality rate was observed. On the other hand, the mortality rate of HUS due to E. coli O104:H4 in this outbreak was 3.74% (WHO, 2011). E. coli O104:H4 appears to be an enteroaggregative E. coli (EAEC) that has acquired the ability to produce Stx2, typically produced by enterohemorrhagic E. coli (EHEC) rather than EAEC group members. This may have occurred via horizontal gene transfer resulting in a new E. coli virotype dubbed the Enteroaggregative Hemorrhagic E. coli or EAHEC (Bloch et al., 2012). The O104:H4 serotype harbors two copies of the Stx2-encoding prophage. Therefore, this emergent bacterium seems to have a rather novel epidemiologic and pathogenic profile (Brzuszkiewicz et al., 2011;Mellmann et al., 2011). While ruminants are the reservoir of most STEC serotypes, no animal reservoir has been identified for E. coli O104:H4 and humans are believed to be the major reservoir for this organism (Wieler et al., 2011;Auvray et al., 2012;Karch et al., 2012). Whereas, the clinical profile of E. coli O104:H4 was relatively similar to that caused by other STEC infections some pertinent differences existed. For instance, about a quarter of subjects affected developed HUS during the 2011 outbreak, which is 2-5 fold higher than the rate usually observed for an STEC infection (WHO, 2011).

Treatment of an STEC Infection
The lack of an effective treatment strategy for an STEC infection has made these agents a prominent public health threat and a burden to the medical community at large. The currently recommended management of an STEC infection mainly relies on supportive therapy and hydration (Thorpe, 2004). The use of antimicrobial agents in treating these infections has been associated with an increased risk of HUS and is therefore contraindicated (Qadri and Kayali, 1998;Guerrant et al., 2001;Safdar et al., 2002).

Novel and Alternative STEC Treatment Strategies
The debatable use of antimicrobial agents for the treatment of an STEC infection has led to the rise of various alternative treatment approaches ( Table 1). These have ranged from the use of natural products to the development of novel regimens that re-examine employing antimicrobials.

Shiga Toxin Receptor Analog
Various agents that mimic Stx receptors and bind them thus reducing their availability to cellular receptors have been developed. Carbosilane dendrimers harboring Gb3 at their termini neutralize Shiga toxins in vitro and were demonstrated to protect challenged mice when administered intravenously (Nishikawa et al., 2002(Nishikawa et al., , 2005. Similarly, multivalent carbohydrate compounds, such as STARFISH and Daisy also neutralize Shiga toxins in vitro and in animals (Kitov et al., 2000;Mulvey et al., 2003). Gb3 polymers with highly clustered trisaccharides bind Shiga toxins with high affinity and protect challenged mice when administered orally (Watanabe et al., 2004). Recombinant bacterial strains that express toxin receptor mimics have also demonstrated a potential efficacy in vitro and upon testing in animals (Paton et al., 2000(Paton et al., , 2001Hostetter et al., 2014). SYNSORB Pk, a synthetic Stx receptor analog consisting of a Pk trisaccharide bound to Chromosorb R P, a multipurpose sorbent medium, was shown to have an abrogative effect on Shiga toxins in vitro. This agent, however, was not effective in clinical trials (Trachtman et al., 2003).

Antibodies
Preparations of antibodies that can bind Shiga toxins and neutralize their effects have been reported. Anti-lipopolysaccharide antibodies have shown protective abilities upon laboratory assessment (Paton et al., 1998) and monocolonal anti-Stx A subunit antibodies have demonstrated potential utility in both laboratory and animal studies (Islam and Stimson, 1990). Bovine colostrum antibodies against Shiga toxins have also been demonstrated to protect challenged animals (Kuribayashi et al., 2006(Kuribayashi et al., , 2009Seita et al., 2013). A bovine colostrum preparation, rich in immunoglobulins and harboring a high titer of anti-Stx1 and anti-Stx2 antibodies, has also been assessed; a colostrumtreated group of 13 patients and 14 placebo-treated controls were compared. The median frequency of stool excretion was decreased in the colostrum-treated patients; however, the presence of the bacterial agent in subject stools was not notably affected. Study subjects were not monitored for the effect of this treatment on the development of HUS or other potential sequelae of infection (Huppertz et al., 1999

Natural Products
Various natural products have been considered as potential therapeutic agents for STEC infections. These have included lactic acid (Pittman et al., 2012), fruit juices (Nogueira et al., 2003) in addition to plant, fruit and root products, teas or extracts (Tomita et al., 1997;Isogai et al., 1998;Okubo et al., 1998;Takahashi et al., 1999;Heredia et al., 2005;Takemasa et al., 2009;Lacombe et al., 2010;Lee and Stein, 2011;Voravuthikunchai et al., 2012;Liu et al., 2013;Pellarin et al., 2013). These products have shown promise in vitro or in experimental animal models; however, they have not been evaluated in clinical studies. Worth noting is a study that showed a synergistic effect between green tea extract and an antibiotic, levofloxacin, in the treatment of an STEC-infected mouse model (Isogai et al., 2001) indicating that a potential risk imparted by an antibiotic treatment may be lessened by the inclusion of another agent.

Antimicrobial Agents
The use of antimicrobial agents in treating STEC infections has been controversial and the subject of an ongoing debate. While some studies indicated that the use of particular agents may increase the risk of HUS, others have reported a decrease of this risk upon implementation of antimicrobials. While these observations may be particular to certain agents at some doses, the potential risk of antimicrobial treatment inducing HUS has led to a general contraindication of such agents (Qadri and Kayali, 1998;Guerrant et al., 2001;Safdar et al., 2002). Antimicrobials are thought to augment the risk of HUS by enhancing the release of Shiga toxins from bacterial cells via a number of ways. DNA damage that can be caused by some antimicrobials may trigger the bacterial SOS response in STEC cells. The SOS response, whose function is to cope with genomic damage, results in the expression of a number of proteins that may activate the lytic cycle of the bacteriophage encoding a Stx thus enhancing its production. Other types of physiologic stresses caused by antimicrobial agents may also trigger the lytic cycle and result in increased toxin expression (Kimmitt et al., 2000;Los et al., 2009). On the other hand, Stx1 is known to be stored within the periplasmic space of STEC cells; therefore, cellular lysis induced by an antimicrobial agent may result in an enhanced release of this particular type of Stx (Strockbine et al., 1986;Yoh et al., 1997;Sato et al., 2003;Shimizu et al., 2009). Several antimicrobial agents have been shown to enhance the release or the production of Shiga toxins from STEC cells in vitro; these include the quinolones, trimethoprim, and furazolidone (Kimmitt et al., 2000); however, observations indicate that these effect may be strain and antimicrobial agent-specific (Grif et al., 1998). For example some isolates of E. coli O104:H4 from the 2011 outbreak in Europe do not display an increase in toxin production upon treatment with meropenem, ciprofloxacin, chloramphenicol, or fosfomycin, unlike E. coli O157:H7 (Corogeanu et al., 2012). Our group assessed the effect of sub-MIC levels of various antimicrobial agents on triggering the SOS response and the production of Shiga toxins in E. coli O157:H7 and in E. coli O104:H4. A sub-MIC concentration may, after all, be the concentration available locally at the site of infection. We noted that the response is variable depending on the isolate used and the concentration of antimicrobial implemented (Nassar et al., 2013;Fadlallah et al., 2015).
Reconsideration of treating STEC infections with antimicrobial agents has nevertheless gained ground in recent years. Ciprofloxacin was recently reported to decrease the risk of HUS in subjects infected with E. coli O104:H4 during the 2011 outbreak (Geerdes-Fenge et al., 2013) and a reduced duration of carriage of the organism in subjects treated with azithromycin was detected during this outbreak as well . Worth noting, however, is that only a small number of treated subjects was included in both studies. Our group assessed the use of rifampicin at a concentration that decreases toxin production, but at which E. coli O157:H7 cells remain viable, followed by treatment with gentamicin at a bactericidal concentration. This strategy was effective in decreasing toxin release compared to solely treating the cells with a bactericidal gentamicin concentration (Kanbar et al., 2003;Matar and Rahal, 2003). Applying a similar strategy in an E. coli O157:H7 infection mouse model resulted in an improved animal survival rate (Rahal et al., 2011a,b). Utilizing the same strategy to treat E. coli O104:H4 infected mice similarly resulted in an improved survival rate compared to untreated control mice that were infected with the organism; however, the highest survival rate observed was with mice treated with gentamicin alone, unlike our observations with E. coli O157:H7 (Fadlallah et al., 2015). This again highlights observations indicating that different STEC serotypes and even isolates of the same serotype respond differently to antimicrobial treatments.

Probiotics, Phages and Vaccines
Although probiotics may not have a therapeutic benefit in the management of an STEC infection, they may have a relevant preventative utility. Probiotics are probably capable of disrupting host-infectious agent/toxin interactions by occupying cellular receptors themselves, by producing decoy receptors that take up the toxins or by modifying the local milieu, hence making these interactions unfavorable (Corr et al., 2009). Multiple studies have shown in vitro beneficial effects of probiotics and that inoculation of animal models with a probiotic prior to an experimental STEC infection has preventative capabilities (Asahara et al., 2004;Reissbrodt et al., 2009;Eaton et al., 2011;Mogna et al., 2012;Chen et al., 2013;Kakisu et al., 2013;Rund et al., 2013;Stanford et al., 2014). The extent of probiotic protective capabilities seen in experimental models is likely dependent on the probiotic strain used and its ability to modify the surrounding medium. For example, the production of acetate by the probiotic agent has been demonstrated to be an important factor (Fukuda et al., 2011(Fukuda et al., , 2012 and the production of butyric acid and lactic acid may be of relevance as well (Ogawa et al., 2001;Takahashi et al., 2004). One in vitro study showed that cultivation of STEC organisms in the presence of various Bifidobacterium, Pediococcus, and Lactobacillus strains results in a decreased production of Stx2. This was attributed to a decrease in pH due to the acids produced by these agents (Carey et al., 2008). Worth noting is the recombinant probiotic agent that can produce toxin receptor mimics described in section 3.a. (Paton et al., 2000(Paton et al., , 2001Hostetter et al., 2014). Also of relevance are the various studies indicating that the administration of probiotic agents to cattle may reduce their carriage of STEC organisms (systematically reviewed in Sargeant et al., 2007), hence effectively reducing the risk of transmitting these toxigenic agents.
Another preventative measure proposed as a means of controlling STEC is the application of lytic phages. Lytic phages have been shown to reduce STEC numbers in vitro (Niu et al., 2009;Rivas et al., 2010), in phage-treated food products (Abuladze et al., 2008;Anany et al., 2011), on hard surfaces (Abuladze et al., 2008), in mice and in some ruminants (Raya et al., 2006;Sheng et al., 2006). Phage-containing products that can be sprayed on animal hides or on meat products for the control of STEC organisms are available on the market and are Food and Drug Administration (FDA) approved (Sillankorva et al., 2012). The efficacy of orally treating cattle with lytic phages, however, was reported to be limited and requires the development of an enhanced approach or delivery mode (Stanford et al., 2010). Bacteriophages used to eradicate STEC agents may also have a therapeutic utility should the safety and efficacy of such an application be demonstrated in humans.
In conclusion, despite the passage of more than three decades since STEC organisms were first associated with human clinical illness (CDC, 1982), a generally-accepted successful therapeutic method for these organisms remains undocumented. Various approaches have nevertheless been attempted including ones that reconsider the implementation of antimicrobial agents; beneficial effects have been reported for some agents with outcomes appearing dependent on the antimicrobials used, their dose and the STEC isolate itself. Further studies examining antimicrobial agents in the therapy of STEC infections should be conducted in animals to select the safest and most efficacious regimen that would then be assessed in clinical trials.