Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) comprise an important group of enteric pathogens causing hemorrhagic colitis and hemolytic uremic syndrome. These bacteria need EspA (E) as a conduct for Tir (T) delivery to the host cell and surface arrayed intimin (I) which docks the bacterium to the translocated Tir. This phenomenon leads to attaching and effacing (A/E) lesions. A trivalent recombinant protein called rEIT composed of immunologically important portions of EspA, Intimin and Tir was constructed as a candidate vaccine. For high-level expression, the EIT gene was synthesized with codon bias of E. coli. The immunization was conducted in mice with purified rEIT. The results showed that this chimeric protein induced strong humoral response as well as protection against live challenges using EHEC.     

Advances in the development of enterohemorrhagic Escherichia coli vaccines using murine models of infection

Enterohemorrhagic Escherichia coli (EHEC) strains are food borne pathogens with importance in public health. EHEC colonizes the large intestine and causes diarrhea, hemorrhagic colitis and in some cases, life-threatening hemolytic-uremic syndrome (HUS) due to the production of Shiga toxins (Stx). The lack of effective clinical treatment, sequelae after infection and mortality rate in humans supports the urgent need of prophylactic approaches, such as development of vaccines. Shedding from cattle, the main EHEC reservoir and considered the principal food contamination source, has prompted the development of licensed vaccines that reduce EHEC colonization in ruminants. Although murine models do not fully recapitulate human infection, they are commonly used to evaluate EHEC vaccines and the immune/protective responses elicited in the host. Mice susceptibility differs depending of the EHEC inoculums; displaying different mortality rates and Stx-mediated renal damage. Therefore, several experimental protocols have being pursued in this model to develop EHEC-specific vaccines. Recent candidate vaccines evaluated include those composed of virulence factors alone or as fused-subunits, DNA-based, attenuated bacteria and bacterial ghosts. In this review, we summarize progress in the design and testing of EHEC vaccines and the use of different strategies for the evaluation of novel EHEC vaccines in the murine model.     

Neutralizing antibodies to Shiga toxin type 2 (Stx2) reduce colonization of mice by Stx2-expressing Escherichia coli O157:H7

Previously, we showed that the Shiga toxin type 2 (Stx2)-expressing Escherichia coli O157:H7 strain 86-24 colonized mice better than did its isogenic stx₂ negative mutant. Here, we confirmed that finding by demonstrating that Stx2 given orally to mice increased the levels of the 86-24 stx₂ mutant shed in feces. Then we assessed the impact of Stx2-neutralizing antibodies, administered passively or generated by immunization with an Stx2 toxoid, on E. coli O157:H7 colonization of mice. We found that such antibodies reduced the E. coli O157:H7 burden in infected mice and, as anticipated, also protected them from weight loss and death.     

Oral immunization with Lactococcus lactis-expressing EspB induces protective immune responses against Escherichia coli O157:H7 in a murine model of colonization

Enterohemorrhagic Escherichia coli (EHEC) have been responsible for several outbreaks of hemolytic–uremic syndrome (HUS) worldwide. HUS is the most common cause of acute renal failure in children and results in fatalities as high as 50% in the elderly. Currently, neither a specific treatment nor a vaccine is available for EHEC. Lactococcus lactis is a generally regarded as safe “GRAS” bacterium that offers a valuable platform for oral vaccine delivery. Toward the development of an oral vaccine against EHEC, we have previously constructed a recombinant L. lactis strain expressing the EHEC antigen, EspB in the cytoplasmic compartment. However, oral immunization of mice with this strain induced weak priming of the immune system. This outcome was attributed to the rather low levels of EspB expressed by this recombinant strain. Therefore, in the present study we optimized the expression of EspB in L. lactis by secreting the antigen either under constitutive or nisin-inducible control. Indeed, oral immunization of mice with the EspB-secreting strains successfully induced specific mucosal and systemic antibody responses. These responses were associated with mixed Th1/Th2 cell responses in Peyer's Patches and mesenteric lymph nodes. Moreover, immunized mice exhibited significant protection against E. coli O157:H7 colonization, as indicated by the reduced amount and/or duration of the bacterial fecal shedding. Our results demonstrate the protective potential of EspB as an oral vaccine against EHEC infection. Additionally, the study demonstrates the efficient delivery of recombinant EspB by the “GRAS” bacterium, L. lactis. The safety profile of L. lactis as a vaccine vehicle can particularly be beneficial to children and elderly as high-risk groups for HUS incidence.     

Carriage of diarrhoeagenic Escherichia coli by older children and adults in Accra, Ghana

Diarrhoeagenic Escherichia coli (DEC) were sought in stool specimens from 72 adults and children aged over 3 years, who presented with diarrhoea at a hospital in Accra, Ghana, and 72 matched controls. Only diffusely-adherent E. coli were significantly associated with disease in these older individuals (P = 0.029). We additionally tested 53 specimens from infants among whom DEC were collectively associated with disease (P = 0.012). Enteropathogenic, enterotoxigenic and enteroaggregative E. coli, the most commonly isolated pathotypes from infants with diarrhoea, were frequently recovered from healthy adults. Asymptomatic carriage of DEC by older individuals in Accra may place young children at risk for diarrhoea.     

Alternatives to carbapenems in ESBL-producing Escherichia coli infections

Objectives

The authors had for objective to assess the activity of a wide panel of antibiotics on extended-spectrum-β-lactamase producing Escherichia coli isolates (ESBL-Ec), because of the sharp increase of their frequency, leading to an increased use of carbapenems.

Material and methods

We selected 100 ESBL-Ec in which ESBLs were identified by PCR and sequencing, between 2009 and 2010. We determined the MICs of amoxicillin-clavulanate, piperacillin-tazobactam, temocillin, mecillinam, cefoxitin, cefotaxime, ceftazidime, aztreonam, tigecycline, nitrofurantoin, and fosfomycin using reference methods. The susceptibility profiles were defined according to EUCAST 2012 recommendations.

Results

Fosfomycin, nitrofurantoin, and pivmecillinam were active against more than 90% of isolates and remain excellent choices for the oral treatment of urinary tract infections (UTIs). Temocillin and piperacillin-tazobactam are also good candidates for the treatment of pyelonephritis or bloodstream infections. Only 27, 23, and 8% of isolates were susceptible to ceftazidime, cefepime, and cefotaxime, respectively.

Conclusion

Our study results prove that in many cases, there are non-carbapenem alternatives for the treatment of ESBL-Ec infections.     

Shopper cards data and storage practices for the investigation of an outbreak of Shiga-toxin producing Escherichia coli O157 infections

Introduction

An outbreak of shiga-toxin producing Escherichia coli infections occurred in southwest France in June 2012. The outbreak was investigated to identify the source of infection, and guide control measures.

Methods

Confirmed outbreak cases were patients who developed bloody diarrhoea or haemolytic uremic syndrome (HUS) between 28 May and 6 July 2012, with E. coli O157 isolates showing indistinguishable patterns on pulse field gel electrophoresis (PFGE). A standardized questionnaire was administered to patients to document food consumption and other risk exposures. Their purchase was checked through their supermarket shopper card data.

Results

Six patients (four with HUS and two with bloody diarrhea) were confirmed outbreak cases. Fresh ground beef burgers from one supermarket were the only common food exposure, identified by interviews and shopper card data. The PFGE profile of shiga toxin-producing E. coli O157 isolated from the suspected beef burgers was identical to those from the human cases. The suspected beef burgers were no longer on sale at the time of investigation but three patients confirmed as outbreak cases had deep-frozen some at home.

Conclusion

Shopper card data was particularly useful to obtain precise and reliable information on the traceability of consumed food. Despite the expired use-by date, a recall was issued for the beef burgers. This contributed to preventing other cases among consumers who had deep-frozen the beef burgers.     

DNA vaccine encoding the major virulence factors of Shiga toxin type 2e (Stx2e)-expressing Escherichia coli induces protection in mice

Piglet edema disease is found worldwide and has historically been treated with antibiotics. However, no commercial vaccines are available for its prevention. In this study, the two major virulence factors of Shiga-toxigenic Escherichia coli (Stx2eB and FedF) were cloned to a pcDNA6.0 plasmid to develop a novel DNA vaccine against piglet edema disease. In animal trial in mouse model, the antibody titer, mortality, serum cytokine levels (interleukin-1 beta, interleukin-6, tumor necrosis factor alpha and C-reactive protein), serum malondialdehyde level and serum total superoxide dismutase activity were measured to validate the effectiveness of the DNA vaccine. The results show that Stx2eB and FedF at least partially protect against edema disease and FedF is more effective than Stx2eB. Co-immunization with both Stx2eB and FedF is most effective for protecting mice from a subsequent challenge with E. coli O139 (which is known to cause edema disease in pigs).     

Epidemiological analysis of a cluster within the outbreak of Shiga toxin-producing Escherichia coli serotype O104:H4 in Northern Germany, 2011

In May 2011 one of the worldwide largest outbreaks of haemolytic uraemic syndrome (HUS) and bloody diarrhoea caused by Shiga toxin-producing Escherichia coli (STEC) serotype O104:H4 occurred in Germany. One of the most affected federal states was Lower Saxony. We present the investigation of a cluster of STEC and HUS cases within this outbreak by means of a retrospective cohort study. After a 70th birthday celebration which took place on 7th of May 2011 among 72 attendants seven confirmed cases and four probable cases were identified, two of them developed HUS. Median incubation period was 10 days. Only 35 persons (48.6%) definitely answered the question whether they had eaten the sprouts that were used for garnishing the salad. Univariable analysis revealed different food items, depending on the case definition, with Odds Ratio (OR) > 1 indicating an association with STEC infection, but multivariable logistic regression showed no increased risk for STEC infection for any food item and any case definition. Sprouts as the source for the infection had to be assumed based on the results of a tracing back of the delivery ways from the catering company to the sprouts producer who was finally identified as the source of the entire German outbreak. In this large outbreak several case–control studies failed to identify the source of infection.     

Vaccination with EtpA glycoprotein or flagellin protects against colonization with enterotoxigenic Escherichia coli in a murine model

Enterotoxigenic Escherichia coli (ETEC) remain a leading cause diarrheal illness, prompting a search for vaccine targets that led to the recent discovery of EtpA, a secreted adhesin of ETEC that acts by bridging flagella and host cells. In a murine model, immunization with recombinant EtpA glycoprotein inhibited colonization by two EtpA-producing human ETEC strains, H10407 and E24377A. In addition, vaccination with recombinant flagellin (serotype H11) generated antibodies that specifically recognized the tips of flagella from E24377A expressing a heterologous flagellar serotype (H28) and afforded significant protection against colonization. EtpA and/or flagellin could be valuable subunit antigens in the formulation of a broadly protective ETEC vaccine.     

Risk factors for quinolone-resistance in women presenting with Escherichia coli acute pyelonephritis

In France, according to the National Epidemiology Observatory of Bacterial Resistance to Antibiotics, 15.3% of outpatient urinary Escherichia coli isolates were fluoroquinolone-resistant in 2010. This puts to question the relevance of empirical fluoroquinolone therapy for community-acquired acute pyelonephritis (APN), potentially severe infections.     

Development of a fast-dissolving tablet formulation of a live attenuated enterotoxigenic E. coli vaccine candidate

Vaccination is considered the most cost-effective approach to preventing infectious diseases, yet better formulations and delivery methods for efficient distribution and administration of vaccines are needed, especially for low-resource settings. A fast-dissolving tablet (FDT) that could be packaged in a compact stackable blister sheet is a potentially attractive option for formulating oral vaccines, since it would minimally impact the cold chain and could potentially be administered directly to patients without reconstitution. This study focused on using one component of a live attenuated trivalent vaccine under development to produce a FDT for the prevention of diarrhea induced by enterotoxigenic Escherichia coli (ETEC). Ten formulations were prepared and freeze dried to produce FDTs. Three freezing conditions were explored, along with different drying and package sealing methods. Physical properties examined included structural integrity, dissolution time, moisture content, and glass transition temperature. Bacterial viability was tested by assaying for colony-forming units. The formulation compositions and freeze-drying parameters were adjusted in an iterative process to arrive at a promising formulation for the ETEC vaccine tablet. This formulation included sucrose and trehalose as cryoprotectants; phosphate and glutamate salts as buffers and stabilizers; and Natrosol^®, polyvinylpyrrolidone, and mannitol as binders. The process loss in viability during freeze drying was less than 0.3 log₁₀ (50% recovery) for the optimized vaccine tablet formulation. The final tablets were robust, disintegrated in less than 10 s, and preserved the bacteria at 2–8 °C for at least 12 months with less than 0.4 log₁₀ loss (40% recovery) in viability during storage. This study indicates that the FDT produced by freeze drying directly in a blister sheet could be a practical option for formulating ETEC vaccines for oral immunization and help to facilitate delivery of lifesaving vaccines, particularly in low-resource settings.     

Capsular polysaccharide and the O-specific antigen impede antibody binding: A potential obstacle for the successful development of an extraintestinal pathogenic Escherichia coli vaccine

Extraintestinal pathogenic Escherichia coli (ExPEC) cause a wide variety of infections that are responsible for significant morbidity, mortality and costs to our healthcare system. An efficacious vaccine against ExPEC would be desirable. Previously, we demonstrated that nasal immunization with a genetically engineered strain in which capsule and O-antigen are no longer expressed (CP923) was immunogenic, generated antibodies that bound a subset of heterologous ExPEC strains, and enhanced neutrophil-mediated bactericidal activity against the homologous and a heterologous strain in vitro. In the work reported here we tested the hypothesis that nasal immunization with CP923 conferred protection in a mouse intravenous sepsis model. Nasal immunization with the wild-type strain CP9 conferred protection against challenge with itself and this protection was enhanced when IL-12 was used as an adjuvant. However, when CP923 was used the immunogen, protection was not observed against challenge with CP9. Next, we hypothesized that the observed lack of protection may be due to capsule and the O-antigen moiety of lipopolysaccharide (LPS) impeding antibody binding to non-capsule and O-antigen epitopes. This hypothesis was substantiated by in vitro binding assays, which demonstrated that binding of polyclonal anti-CP923 antisera was decreased when capsule and/or O-antigen were present. Lastly, neutrophil-mediated bactericidal activity against CP923, opsonisized with anti-CP923 antisera, was significantly increased compared to CP9. Taken together, these results demonstrate that the capsule and O-antigen form a biologically significant barrier against antibodies directed against non-capsular and O-antigen epitopes. This defense against the acquired immune response will need to be overcome for the development of a successful vaccine against ExPEC.     

Vaccination of attenuated EIS-producing Salmonella induces protective immunity against enterohemorrhagic Escherichia coli in mice

There is an urgent need for vaccine against enterohemorrhagic Escherichia coli (EHEC), which causes a wide range of life-threatening diseases in human and animals. E. coli secreted protein A (EspA), intimin and shiga toxin (Stx) are important pathogenic factors and protective antigens of EHEC. In our previous study, we found that recombinant trivalent protein EIS, which is composed of EspA (E), the 300 amino acids of the carboxyl terminus of intimin (I) and the B subunit of Stx2 (S), was able to efficiently elicit protective immunity against EHEC. The application of live attenuated Salmonella as a carrier for vaccine against mucosal pathogens provided unparalleled merits. Therefore, in this study we constructed live attenuated EIS-producing Salmonella vaccine and tested it as vaccine in mice model. We found that the vaccination of EIS-producing recombinant Salmonella was able to induce significant increases of EspA, intimin and Stx2 specific IgG in serum and secretory IgA in feces. Antigen specific T cell proliferation was also observed in the mice immunized with recombinant EIS-producing Salmonella. In addition, this immunity was able to protect mice from a challenge of a lethal dose of EHEC, even after a period of 70 days. Moreover, the EIS-producing Salmonella induced immunity can be boosted by a single subcutaneous injection of purified EIS protein, even after an interval of 70 days. This EIS-producing Salmonella vaccine provides an alternative approach for the prevention of EHEC infection.     

Immunogenicity of a novel Stx2B–Stx1B fusion protein in a mice model of Enterohemorrhagic Escherichia coli O157:H7 infection

Shiga toxin type 1 and 2 produced by Enterohemorrhagic Escherichia coli (EHEC) O157:H7 are responsible for hemolytic uremic syndrome, a life-threatening sequela. We constructed a novel fusion protein carrying both of the immunogenic B subunits derived from the two toxins, designated Stx2B–Stx1B (2S for short), expressed in the E. coli BL21 and harvested the purified protein by a simple anion-exchange chromatography method. The fusion protein induced high level humoral IgG in mice, subclass analysis showed IgG1 dominate the IgG increase trend, which indicated that a partial to Th2 response contributed to this humoral reactivity. High level neutralizing antibodies elicited by this fusion protein inhibited cytotoxicity of toxins and protected mice from lethal dose challenge of lysed EHEC O157:H7.     

Engineering of Escherichia coli strains for plasmid biopharmaceutical production: Scale-up challenges

Plasmid-based vaccines and therapeutics have been making their way into the clinic in the last years. The existence of cost-effective manufacturing processes capable of delivering high amounts of high-quality plasmid DNA (pDNA) is essential to generate enough material for trials and support future commercialization. However, the development of pDNA manufacturing processes is often hampered by difficulties in predicting process scale performance of Escherichia coli cultivation on the basis of results obtained at lab scale. This paper reports on the differences observed in pDNA production when using shake flask and bench-scale bioreactor cultivation of E. coli strains MG1655ΔendAΔrecA and DH5α in complex media with 20 g/L of glucose. MG1655ΔendAΔrecA produced 5-fold more pDNA (9.8 mg/g DCW) in bioreactor than in shake flask (1.9 mg/g DCW) and DH5α produced 4-fold more pDNA (8 mg/g DCW) in bioreactor than in shake flask (2 mg/g DCW). Accumulation of acetate was also significant in shake flasks but not in bioreactors, a fact that was attributed to a lack of control of pH.     

Source identification of airborne Escherichia coli of swine house surroundings using ERIC-PCR and REP-PCR

Evidence is mounting that microorganisms originating from livestock impact the air quality of the animal houses themselves and the public in the surrounding neighborhoods. The aim of this study was to develop efficient bacterial source tracking capabilities to identify sources of Escherichia coli aerosol pollution caused by pigs. Airborne E. coli were isolated from indoor air, upwind air (10 and 50 m away) and downwind air samples (10, 50, 100, 200 and 400 m away) for five swine houses using six-stage Andersen microbial samplers and Reuter-Centrifugal samplers (RCS). E. coli strains from pig fecal samples were also collected simultaneously. The enterobacterial repetitive intergenic consensus polymerize chain reaction (ERIC-PCR) and the repetitive extragenic palindromic (REP-PCR) approaches were used to study the genetic variability and to determine the strain relationships among E. coli isolated from different sites in each swine house. Results showed that 35.1% (20/57) of the bacterial DNA fingerprints from the fecal isolates matched with the corresponding strains isolated from indoor and downwind air samples (similarity ?90%). E. coli strains from the indoor and downwind air samples were closely related to the E. coli strains isolated from feces, while those isolated from upwind air samples (swine house C) had low similarity (61-69%). Our results suggest that some strains isolated from downwind and indoor air originated in the swine feces. Effective hygienic measures should be taken in animal farms to prevent or minimize the downwind spread of microorganism aerosol.     

Toward the development of a stable,freeze-dried formulation of Helicobacter pylori killed whole cell vaccine adjuvanted with a novel mutant of Escherichia coli heat-labile toxin

No vaccine exists for the prevention of infection with the ubiquitous gastric pathogen Helicobacter pylori, and drug therapy for the infection is complicated by poor patient compliance, the high cost of treatment, and ineffectiveness against drug-resistant strains. A new medical advancement is required to reduce the incidence of peptic ulcer disease and stomach cancer, two conditions caused by infection with H. pylori. Clinical trials have been performed with a formalin-inactivated H. pylori whole cell (HWC) vaccine, given orally in combination with the mucosal adjuvant mLT(R192G), a mutant of Escherichia coli heat-labile toxin. Following the initial dose of this vaccine, some subjects experienced gastrointestinal side effects. To reduce side effects and potentially further increase the amount of adjuvant that can safely be administered with the HWC vaccine, experiments were performed with a form of LT that carried two mutations in the A subunit, a substitution of G for R at position 192, and A for L at position 211. The double mutant LT (dmLT) adjuvant stimulated immune responses as effectively as the single mutant LT in mice. Additionally, following a challenge infection, the dmLT-adjuvanted vaccine was as effective as single mutant LT in reducing gastric urease levels (diagnostic for H. pylori infection), and H. pylori colonization in the stomach as assessed by quantitative analysis of stomach homogenates. A lyophilized formulation of HWC was developed to improve stability and to potentially reduce reliance on cold chain maintenance. It was observed that a dmLT-adjuvanted lyophilized vaccine was equally as protective in the mouse model as the liquid formulation as assessed by gastric urease analysis and analysis of stomach homogenates for viable H. pylori. No readily detectable effect of tonicity or moisture content was observed for the lyophilized vaccine within the formulation limits evaluated. In an accelerated stability study performed at 37 °C the lyophilized vaccine remained equally as protective as vaccine stored at 2–8 °C. The formulation selected for clinical development consisted of 2.5 × 10¹⁰ formalin-inactivated cells per ml in 6.5% trehalose, 0.5% mannitol, and 10 mM citrate buffer at pH 6.8.     

Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen that can cause gastrointestinal disease with potentially fatal consequences as a result of systemic Shiga toxin activity. Cattle are the main reservoir host of EHEC O157 and interventions need to be developed that prevent cattle colonization or limit shedding of the organism from this host. EHEC O157 predominately colonizes the bovine terminal rectum and requires a type III secretion system (T3SS) for adherence and persistence at this site. A vaccine based on concentrated bacterial supernatant that contains T3S proteins has shown some efficacy. Here we have demonstrated that vaccination with a combination of antigens associated with T3S-mediated adherence; the translocon filament protein, EspA, the extracellular region of the outer membrane adhesin, intimin, and the translocated intimin receptor (Tir) significantly reduced shedding of EHEC O157 from experimentally infected animals. Furthermore, this protection may be augmented by addition of H7 flagellin to the vaccine preparation that has been previously demonstrated to be partially protective in cattle. Protection correlates with systemic and mucosal antibody responses to the defined antigens and validates the targeting of these colonization factors.