Formulation Studies to Develop Low-Cost, Orally-Delivered Secretory IgA Monoclonal Antibodies for Passive Immunization Against Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) is a common cause for diarrheal infections in children in low- and middle-income countries (LMICs). To date, no ETEC vaccine candidates have been approved. Passive immunization with low-cost, oral formulations of secretory IgA (sIgA) against ETEC is an alternative approach to protect high-risk populations in LMICs. Using a model sIgA monoclonal antibody (anti-LT sIgA2-mAb), the stability profiles of different formulations were assessed during storage and in in vitro digestion models (mimicking in vivo oral delivery). First, by employing various physicochemical techniques and a LT-antigen binding assay, three formulations with varying acid-neutralizing capacity (ANC) were evaluated to stabilize sIgA2-mAb during stress studies (freeze-thaw, agitation, elevated temperature) and during exposure to gastric phase digestion. Next, a low-volume, in vitro intestinal digestion model was developed to screen various additives to stabilize sIgA2-mAb in the intestinal phase. Finally, combinations of high ANC buffers and decoy proteins were assessed to collectively protect sIgA2-mAb during in vitro sequential (stomach to intestine) digestion. Based on the results, we demonstrate the feasibility of low-cost, 'single-vial', liquid formulations of sIgA-mAbs delivered orally after infant feeding for passive immunization, and we suggest future work based on a combination of in vitro and in vivo stability considerations.

Assessment the role of some Bacillus strains in improvement rex rabbits resistance against ETEC challenge.

Increasing reports have indicated that specific strains of probiotic Bacillus have the potential to prevent diseases. The purpose of this study was to explore the effects of three Bacillus strains (Bacillus subtilis BSWJ2017001, Bacillus pumilus BSWJ2017002, and B. subtilis BSWJ2017003) mixture dietary supplementation on rex rabbits infected with enterotoxigenic Escherichia coli (ETEC). In this study, 60 35-day-old weaning rex rabbits were separated into two groups randomly: control group (fed basal diet with no antibiotics) and Bacillus strains group (fed basal diet containing 1.0 × 106 CFU/g Bacillus strains mixture). After 8 weeks of feeding, the rex rabbits were inoculated orally with 5.0 mL of ETEC (1.0 × 109 CFU/mL) and assessed at 0, 12, and 24 h. The Bacillus strains mixture attenuated the oxidative damage, diarrhea severity, and intestinal damage of ETEC infected rabbits. It also significantly increased the population of Lactobacillus spp., and Bifidobacterium spp., and decreased the population of Enterococcus spp.. Moreover, Bacillus strains group exhibited higher levels of toll-like receptor (TLR) 2, anti-inflammatory cytokines, secretory immunoglobulin A, and intestinal barrier-related genes than control group, as well as lower levels of TLR-4 and pro-inflammatory cytokines. These results demonstrated that Bacillus strains mixture could attenuate injury caused by ETEC and enhance disease resistance by improving specific intestinal microbiota members and immunity in weaning rex rabbits.

Recombinant Escherichia coli Cell Lysates as a Low-Cost Alternative for Vaccines Against Veterinary Clostridial Diseases.

This chapter describes a practical, industry-friendly, and efficient vaccine protocol based on the use of Escherichia coli cell fractions (inclusion bodies or cell lysate supernatant) containing the recombinant antigen. This approach was characterized and evaluated in laboratory and farm animals by the seroneutralization assay in mice, thereby showing to be an excellent alternative to induce a protective immune response against clostridial diseases.

In Vitro Characterization and In Vivo Efficacy Assessment in Galleria mellonella Larvae of Newly Isolated Bacteriophages against Escherichia coli K1.

Extra-intestinal Escherichia coli express several virulence factors that increase their ability to colonize and survive in different localizations. The K1 capsular type is involved in several infections, including meningitis, urinary tract, and bloodstream infections. The aims of this work were to isolate, characterize, and assess the in vivo efficacy of phages targeting avian pathogenic E. coli (APEC) O18:K1, which shares many similarities with the human strains responsible for neonatal meningitis. Eleven phages were isolated against APEC O18:K1, and four of them presenting a narrow spectrum targeting E. coli K1 strains were further studied. The newly isolated phages vB_EcoS_K1-ULINTec2 were similar to the Siphoviridae family, and vB_EcoP_K1-ULINTec4, vB_EcoP_K1-ULINTec6, and vB_EcoP_K1-ULINTec7 to the Autographiviridae family. They are capsular type (K1) dependent and present several advantages characteristic of lytic phages, such as a short adsorption time and latent period. vB_EcoP_K1-ULINTec7 is able to target both K1 and K5 strains. This study shows that these phages replicate efficiently, both in vitro and in vivo in the Galleria mellonella model. Phage treatment increases the larvae survival rates, even though none of the phages were able to eliminate the bacterial load.

In silico analysis and in vivo assessment of a novel epitope-based vaccine candidate against uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are common pathogens in urinary tract infections (UTIs), which show resistance to antibiotics. Therefore, there is a need for a vaccine to reduce susceptibility to the infection. In the present study, bioinformatics approaches were employed to predict the best B and T-cell epitopes of UPEC virulence proteins to develop a multiepitope vaccine candidate against UPEC. Then, the efficacy of the candidate was studied with and without Freund adjuvant. Using bioinformatics methods, 3 epitope-rich domains of IutA and FimH antigens were selected to construct the fusion. Molecular docking and Molecular dynamics (MD) simulation were employed to investigate in silico interaction between designed vaccine and Toll-like receptor 4 (TLR4). Our results showed that the levels of IgG and IgA antibodies were improved in the serum and mucosal samples of the vaccinated mice, and the IgG responses were maintained for at least 6 months. The fusion protein was also able to enhance the level of cytokines IFN.γ (Th1), IL.4 (Th2), and IL.17. In challenge experiments, all vaccine combinations showed high potency in the protection of the urinary tract even after 6 months post first injection. The present study indicates that the designed candidate is able to evoke strong protective responses which warrant further studies.

Assessment of extended-spectrum ß-lactamase (ESBLs) - producing Escherichia coli from minced meat of cattle and swab samples and hygienic status of meat retailer shops in Jimma town, Southwest Ethiopia.

BACKGROUND: The impact of animals sources of food as a possible reservoir for extended-spectrum ß-lactamase (ESBL) - Producing E. coli, and the dissemination of such strains into the food production chain need to be assessed. This study was aimed to assess the presence and antimicrobial susceptibility patterns of ESBLs - producing E. coli isolates from minced meat and environmental swab samples at meat retailer shops of Jimma town, Southwest Ethiopia. METHODOLOGY: A cross-sectional descriptive study was conducted from March to June, 2016. A total of 168 minced meat and swab samples were first enriched by buffered peptone water (BPW) for overnight and streaked onto MacConkey agar. Double disk synergy (DDS) method was used for detection of ESBL-producing strains. A disk of amoxicillin + clavulanic acid (20/10 µg) was placed in the center of Mueller-Hinton agar plate, and cefotaxime (30 µg) and ceftazidime (30 µg) were placed at a distance of 20 mm from the central disk. Checklist was used to assess hygienic status of butcher shops and practices meat handlers. RESULTS: A total of 35 (20.80%) biochemically confirmed E. coli were obtained from 168 samples. Of these, 21 (23.9%) of them were detected from 88 minced meat and the other 14 (17.5%) from 80 swab samples taken from butcher's hand, knives, chopping board and protective clothing. From 35 E. coli isolates, 7(20%) of them were confirmed as ESBL- producers. Among ESBL- producing strains, 85.7% were resistant for cefotaxime and ceftriaxone and 71.4% for ceftazidime. Among non-ESBLs-producing strains only seven isolates were resistant to third generation cephalosporin. All E. coli isolates were resistant to ampicillin, penicillin and erythromycin, and susceptible to amikacin. Poor hygienic status of butcher shops and unhygienic practice of meat handlers were observed. CONCLUSION: The detections of ESBLs- producing strains could be contributed for the increment of multi drug resistant isolates. This study also concluded that, unhygienic meat handling and processing practice can contribute for contaminations of meat. Thus, strategies should be planned and implemented to improve the knowledge and practice of butchers about handling and processing of meat.

Implications and measurement of herd protection (indirect effects) for enteric vaccine development.

Diarrhea remains one of the top five causes of disease and death among young children in developing nations. Fortunately, scientists are making progress developing vaccines against enterotoxigenic E. coli (ETEC) and Shigella, two of the leading diarrhea pathogens. As vaccine developers start to consider field efficacy trials of these vaccines, they should be aware of the importance of evaluating not only vaccine direct effects on the immunized, but also the herd effects that vaccination can afford to the unimmunized in a community. In a workshop held at the conference titled "Vaccines against Shigella and ETEC (VASE)", we described to participants what herd effects are and we presented on methods used in cholera and rotavirus studies that could be useful for future ETEC and Shigella vaccine trials conducted in low and middle-income nations. We also presented evidence on the effects of vaccine herd effects for estimates of vaccine cost-effectiveness.

Using advocacy to increase investment in enteric vaccine development.

Catalyzing and sustaining momentum for long-term research investments can be a challenge, especially for enteric pathogens like ETEC andShigella that are most threatening to the health of children in low-resource areas, and whose vaccines would not be for global use. The 2018 Vaccines Against Shigella and ETEC (VASE) Conference included a workshop focused on building the capacity of scientists to communicate about their own research and advocate for additional attention and funding for enteric disease and vaccines research. Workshop presenters shared best practices and examples of advocacy, communications, and messaging tactics that have been used successfully during early stages of vaccine development research for other pathogens. The presentations were followed by an interactive, hands-on training for real-life communication opportunities for scientists that could result in increased research funding, including developing resonant messaging for relevant audiences and practicing interviews.

[Microorganisms responsible of nosocomial infections in the Mexican Social Security Institute]. / Los microorganismos causantes de infecciones nosocomiales en el Instituto Mexicano del Seguro Social.

BACKGROUND: The prevention and control of the nosocomial infections involve the knowledge of the most frequent type of microorganism isolated. In Mexico there are not national statistics to identify the main microorganisms that cause a nosocomial infection. METHODS: It was conducted an analysis of all the culture results of the nosocomial infections reported by the Sistema de Vigilancia Epidemiológica Hospitalaria (Epidemiological Surveillance System) of the Instituto Mexicano del Seguro Social during the year 2013. The most frequent and relevant microorganisms were reported. RESULTS: 48 377 results from cultures of nosocomial infections were studied. Of these 13 207 (27.3 %) were from 25 high specialty medical units and 35 170 (72.6 %) from 197 second level medical units. The most frequently isolated microorganism was Escherichia coli with 8192 results (16.9 %), followed by the group of Coagulase-negative Staphylococci with 6771 cultures (14 %) and Pseudomonas aeruginosa with 5275 results (19.9 %). Slight differences between levels of care and specialized hospitals were observed. CONCLUSION: This study identifies the Escherichia coli, the group of Coagulase-negative Staphylococci and the Pseudomonas aeruginosa as the main microorganisms to prevent.

Introducción: la prevención y el control de las infecciones nosocomiales requiere el conocimiento del tipo de microorganismo que es más frecuentemente aislado. En México se carece de una estadística nacional que identifique el principal microorganismo causante de infecciones nosocomiales. Métodos: se estudiaron todos los resultados positivos de los cultivos de las infecciones nosocomiales reportadas por el sistema de Vigilancia Epidemiológica Hospitalaria del Instituto Mexicano del Seguro Social durante el año 2013. Se reportaron los microorganismos más frecuentes y los de mayor relevancia epidemiológica. Resultados: se estudiaron 48 377 resultados de cultivos de infecciones nosocomiales; de estos, 13 207 (27.3 %) correspondieron a las 25 unidades médicas de alta especialidad y 35 170 (72.6 %) a las 197 unidades médicas de segundo nivel. El microorganismo más frecuentemente aislado fue la Escherichia coli con 8192 (16.9 %), seguido del grupo de los Staphylococcus coagulasa-negativos con 6771 (14 %) y la Pseudomonas aeruginosa 5275 (19.9 %). Se observaron diferencias ligeras entre los niveles de atención y entre los hospitales monotemáticos. Conclusiones: el presente estudio identifica a la Escherichia coli, a los Staphylococcus coagulasa-negativos y a la Pseudomonas aeruginosa como los principales microorganismos que se deben combatir.

Assessment of Shiga Toxin-Producing Escherichia coli O157 Illnesses Prevented by Recalls of Beef Products.

Beef product recall data from 2005 through 2012 associated with Shiga toxin-producing Escherichia coli (STEC) O157 contamination were used to develop quantitative models to estimate the number of illnesses prevented by recalls. The number of illnesses prevented was based on the number of illnesses that occurred relative to the number of pounds consumed, then extrapolated to the number of pounds of recalled product recovered. A simulation using a Program Evaluation and Review Technique (PERT) probability distribution with illness-related recalls estimated 204 (95% credible interval, 117-333) prevented STEC O157 illnesses from 2005 through 2012. Recalls not associated with illnesses had more recalled product recovered and prevented an estimated 83 additional STEC O157 illnesses. Accounting for underdiagnosis resulted in an estimated total of 7500 STEC O157 illnesses prevented over 8 years. This study demonstrates that recalls, although reactive in nature, are an important tool for averting further exposure and illnesses.

Effect of Sampling Plans on the Risk of Escherichia coli O157 Illness.

Australia exports about 150,000 to 200,000 tons of manufacturing beef to the United States annually. Each lot is tested for Escherichia coli O157 using the N-60 sampling protocol, where 60 small pieces of surface meat from each lot of production are tested. A risk assessment of E. coli O157 illness from the consumption of hamburgers made from Australian manufacturing meat formed the basis to evaluate the effect of sample size and amount on the number of illnesses predicted. The sampling plans evaluated included no sampling (resulting in an estimated 55.2 illnesses per annum), the current N-60 plan (50.2 illnesses), N-90 (49.6 illnesses), N-120 (48.4 illnesses), and a more stringent N-60 sampling plan taking five 25-g samples from each of 12 cartons (47.4 illnesses per annum). While sampling may detect some highly contaminated lots, it does not guarantee that all such lots are removed from commerce. It is concluded that increasing the sample size or sample amount from the current N-60 plan would have a very small public health effect.

Potential Impact of Food Safety Vaccines on Health Care Costs.

Foodborne pathogens continue to cause several outbreaks every year in many parts of the world. Among the bacterial pathogens involved, Shiga toxin-producing Escherichia coli, Campylobacter jejuni, and nontyphoidal Salmonella species cause a significant number of human infections worldwide, resulting in a huge annual economic burden that amounts to millions of dollars in health care costs. Human infections are primarily caused by the consumption of contaminated food. Vaccination of food-producing animals is an attractive, cost-effective strategy to lower the levels of these pathogens that will ultimately result in a safer food supply and fewer human infections. However, producers are often reluctant to routinely vaccinate animals against these pathogens since they do not cause any detectable clinical symptoms. This review highlights recent approaches used to develop effective food safety vaccines and the potential impact these vaccines might have on health care costs.

First step in using molecular data for microbial food safety risk assessment; hazard identification of Escherichia coli O157:H7 by coupling genomic data with in vitro adherence to human epithelial cells.

The potential for using whole genome sequencing (WGS) data in microbiological risk assessment (MRA) has been discussed on several occasions since the beginning of this century. Still, the proposed heuristic approaches have never been applied in a practical framework. This is due to the non-trivial problem of mapping microbial information consisting of thousands of loci onto a probabilistic scale for risks. The paradigm change for MRA involves translation of multidimensional microbial genotypic information to much reduced (integrated) phenotypic information and onwards to a single measure of human risk (i.e. probability of illness). In this paper a first approach in methodology development is described for the application of WGS data in MRA; this is supported by a practical example. That is, combining genetic data (single nucleotide polymorphisms; SNPs) for Shiga toxin-producing Escherichia coli (STEC) O157 with phenotypic data (in vitro adherence to epithelial cells as a proxy for virulence) leads to hazard identification in a Genome Wide Association Study (GWAS). This application revealed practical implications when using SNP data for MRA. These can be summarized by considering the following main issues: optimum sample size for valid inference on population level, correction for population structure, quantification and calibration of results, reproducibility of the analysis, links with epidemiological data, anchoring and integration of results into a systems biology approach for the translation of molecular studies to human health risk. Future developments in genetic data analysis for MRA should aim at resolving the mapping problem of processing genetic sequences to come to a quantitative description of risk. The development of a clustering scheme focusing on biologically relevant information of the microbe involved would be a useful approach in molecular data reduction for risk assessment.

An analysis of cattle farmers' perceptions of drivers and barriers to on-farm control of Escherichia coli O157.

Structural equation modelling and survey data were used to test determinants' influence on farmers' intentions towards Escherichia coli O157 on-farm control. Results suggest that farmers more likely to show willingness to spend money/time or vaccinate to control Escherichia coli O157 are those: who think farmers are most responsible for control; whose income depends more on opening farms to the public; with stronger disease control attitudes; affected by outbreaks; with better knowledge and more informed; with stronger perceptions of biosecurity measures' practicality; using a health plan; who think farmers are the main beneficiaries of control; and whose farms are dairy rather than beef. The findings might suggest that farmers may implement on-farm controls for E. coli O157 if they identify a clear hazard and if there is greater knowledge of the safety and efficacy of the proposed controls.

Assessment of the effect of chemical agents used in dentistry on the removal of Porphyromonas gingivalis and Escherichia coli from sandblasted acid-etched titanium dental implants: an in vitro study.

PURPOSE: The aim of this study was to evaluate the capability of chemicals to decontaminate Escherichia coli (E coli) or Porphyromonas gingivalis (P gingivalis) from sandblasted acid-etched (SAE) titanium dental implants. MATERIALS AND METHODS: SAE titanium dental implants were contaminated with E coli or P gingivalis and incubated in a sterile bacterial culture media under aerobic and anaerobic conditions, respectively. The implants were treated with 10 different conditions: calcium hydroxide [Ca(OH)2] paste for 1 minute and saline irrigation for 1 minute; Ca(OH)2 paste for 1 minute and 0.2% chlorhexidine digluconate (CHXD) irrigation for 1 minute; 0.2% CHXD for 1 minute; Dakin's solution for 1 minute; tetracycline hydrochloride (T-HCl) as a 1 g per 20 mL solution for 1, 2, and 3 minutes; and T-HCl paste for 1, 2, and 3 minutes. All implants were irrigated with 1 mL of saline solution and incubated under aerobic and anaerobic conditions for 24 hours or 48 hours for E coli- and P gingivalis-contaminated implants, respectively. The control group was submitted to all procedures except for the chemical treatments. Aliquots were removed, and turbidity was measured by spectrophotometry. The level of bacterial growth in control cultures was considered to have a decontamination percentage (DC%) of 0. RESULTS: Spectrophotometric analysis showed that all chemical treatments resulted in significantly higher DC% compared to controls for SAE implants contaminated with E coli (P < .05) or P gingivalis (P < .05). For the P gingivalis experiments, SAE implants treated with Ca(OH)2 paste and saline solution had a lower DC% (39.3%) than those in the other treatment groups. In the E coli experiments, DC% was significantly lower for SAE implants treated with Ca(OH)2 paste and saline solution (48.7%), Dakin's solution (92.7%), or T-HCl paste for 1 minute (96.6%) than those in the other groups. CONCLUSION: The DC% of SAE implants contaminated with E coli or P gingivalis by means of chemicals commonly used in dentistry is high, with the exception of Ca(OH)2 paste burnished for 1 minute and then irrigated with saline solution for 1 minute.

An assessment of enterotoxigenic Escherichia coli and Shigella vaccine candidates for infants and children.

Despite improvements to water quality, sanitation, and the implementation of current prevention and treatment interventions, diarrhea remains a major cause of illness and death, especially among children less than five years of age in the developing world. Rotavirus vaccines have already begun making a real impact on diarrhea, but several more enteric vaccines will be necessary to achieve broader reductions of illness and death. Among the many causes of diarrheal disease, enterotoxigenic Escherichia coli (ETEC) and Shigella are the two most important bacterial pathogens for which there are no currently licensed vaccines. Vaccines against these two pathogens could greatly reduce the impact of disease caused by these infections. This review describes the approaches to ETEC and Shigella vaccines that are currently under development, including a range of both cellular and subunit approaches for each pathogen. In addition, the review discusses strategies for maximizing the potential benefit of these vaccines, which includes the feasibility of co-administration, consolidation, and combination of vaccine candidates, as well as issues related to effective administration of enteric vaccines to infants. Recent impact studies indicate that ETEC and Shigella vaccines could significantly benefit global public health. Either vaccine, particularly if they could be combined together or with another enteric vaccine, would be an extremely valuable tool for saving lives and promoting the health of infants and children in the developing world, as well as potentially providing protection to travelers and military personnel visiting endemic areas.

Relative cost-effectiveness of a norovirus vaccine in the deployed military setting compared to a vaccine against Campylobacter sp., ETEC, and Shigella sp.

Norovirus (NoV) has been identified as a significant cause of acute gastrointestinal illness among deployed military troops. We conducted a cost-effectiveness analysis for the use of a NoV vaccine in the military using a previously developed model that evaluated vaccines for ETEC, Campylobacter, and Shigella for prevention of non-outbreak associated travelers' diarrhea. Under conservative assumptions, acquisition of a NoV vaccine by the Department of Defense is estimated to result in a cost-effectiveness ratio per duty day lost to illness (CERDDL) of $1344 compared to a CERDDL of $776, $800, and $1275 for ETEC, Campylobacter sp., and Shigella sp., respectively compared to current management strategies. The absolute value of avoiding a duty day lost is likely to vary under different scenarios, and further study is needed to evaluate how improved diagnostics and prevention of outbreaks may impact the relative value of this vaccine. Overall, this study demonstrates the utility of a previously established evidence-based decision tool for prioritization of vaccine acquisition in an important target population.

The evidentiary value of challenge trials for three pre-harvest food safety topics: a systematic assessment.

Reducing zoonotic pathogens in food animals prior to harvest will reduce the pathogen burden that enters the food chain and the environment. Consequently, the burden of enteric illness in humans may be reduced. Evaluating interventions to reduce a pathogen in animals often begins with challenge trials, in which animals are deliberately exposed to the pathogen under controlled conditions. Challenge trials are subsequently followed by field trials, also known as randomized controlled trials, in which the animals are naturally exposed to the pathogen. Challenge trials can most effectively inform field trials only if they precede field trials, are robust, internally valid and transparently reported. Using systematic review and meta-analysis methodology, we examined the pre-harvest food safety literature for three intervention-pathogen-species combinations: probiotics/competitive exclusion products in ruminants to reduce Escherichia coli O157 shedding, vaccines in ruminants to reduce E. coli O157 shedding and vaccines in swine to reduce Salmonella shedding. We examined two outcomes, prevalence of faecal shedding at the end of the trial and prevalence of faecal shedding throughout the trial period, to compare challenge trials and field trials. We found that challenge trials occurred concurrently with field trials, challenge trials suffered from reporting deficiencies of methodological features, challenge trials tended to report a more favourable outcome than field trials, and there was some evidence of publication bias among all three intervention-pathogen-species combinations. Challenge trials would better serve to inform field trials if they precede field trials, are methodologically sound, include transparent reporting and are published regardless of their results. In addition, due to our findings of greater efficacy reported among challenge trials compared with field trials, risk models predicting the public health benefits of pre-harvest interventions to reduce zoonotic pathogens in livestock might be best served by field trial results alone.

[After the crisis is before the crisis: Coping-strategies for public health emergency situations on different levels--experiences of the German E. coli outbreak 2011]. / Nach der Krise ist vor der Krise. Allgemeine Strategien für die erfolgreiche Bewältigung medizinischer Krisensituationen basierend auf den Erfahrungen des EHEC-Ausbruchs 2011.

The German E. coli crisis in 2011 posed multifaceted challenges to the actors of the German health care system. This paper analyzes the outbreak from a multidisciplinary perspective. It offers best practices and general recommendations for future public health emergencies for hospitals as well as at the federal and local level, addressing medical practitioners as well as health care managers.