Enterotoxigenic Escherichia coli (ETEC) infections have been identified as a major cause of acute diarrhoea in children in developing countries, associated with substantial morbidity and mortality rates. Additionally, ETEC remains the most common cause of acute diarrhea of international travellers to endemic areas. The heat-labile toxin (LT) is a major virulence factor of ETEC, with a significant correlation between the presence of antibodies against LT and protection in infected patients. In the present work, we constructed a recombinant LTB unit (rLTB) and studied the capacity of this toxoid incorporated in microneedles (rLTB-MN) to induce a specific immune response in mice. MN were prepared from aqueous blends of the polymer Gantrez AN® [poly (methyl vinyl ether-co-maleic anhydride)], which is not cytotoxic and has been shown to possess immunoadjuvant properties. The mechanical and dissolution properties of rLTB-MNs were evaluated in an in vitro Parafilm M® model and in mice and pig skin ex vivo models. The needle insertion ranged between 378 µm and 504 µm in Parafilm layers, and MNs fully dissolved within 15 min of application inside porcine skin. Moreover, female and male BALB/c mice were immunized through ear skin with one single dose of 5 ¿g·rLTB in MNs, eliciting significant fecal anti-LT IgA antibodies, higher in female than in male mice. Moreover, we observed an enhanced production of IL-17A by spleen cells in the immunized female mice, indicating a mucosal non-inflammatory and neutralizing mediated response. Further experiments will now be required to validate the protective capacity of this new rLTB-MN formulation against this deadly non-vaccine-preventable disease.

Enterotoxigenic Escherichia coli (ETEC) represents a major cause of morbidity and mortality in the human population. In particular, ETEC infections affect children under the age of five from low-middle income countries. However, there is no licensed vaccine against this pathogen. ETEC vaccine development is challenging since this pathotype expresses a wide variety of antigenically diverse virulence factors whose genes can be modified due to ETEC genetic plasticity. To overcome this challenge, we propose the use of outer membrane vesicles (OMVs) isolated from two ETEC clinical strains. In these OMVs, proteomic studies revealed the presence of important immunogens, such as heat-labile toxin, colonization factors, adhesins and mucinases. Furthermore, these vesicles proved to be immunogenic after subcutaneous administration in BALB/c mice. Since ETEC is an enteropathogen, it is necessary to induce both systemic and mucosal immunity. For this purpose, the vesicles, free or encapsulated in zein nanoparticles coated with a Gantrez(R)-mannosamine conjugate, were administered orally. Biodistribution studies showed that the encapsulation of OMVs delayed the transit through the gut. These results were confirmed by in vivo study, in which OMV encapsulation resulted in higher levels of specific antibodies IgG2a. Further studies are needed to evaluate the protection efficacy of this vaccine approach.

Currently, cancer, leishmaniasis and bacterial infections represent a serious public health burden worldwide. Six cinnamyl and benzodioxyl derivatives incorporating selenium (Se) as selenocyanate, diselenide, or selenide were designed and synthesized through a nucleophilic substitution and/or a reduction using hydrides. Ferrocene was also incorporated by a Friedel-Crafts acylation. All the compounds were screened in vitro for their antiproliferative, antileishmanial, and antibacterial properties. Their capacity to scavenge free radicals was also assessed as a first approach to test their antioxidant activity. Benzodioxyl derivatives 2a-b showed cytotoxicity against colon (HT-29) and lung (H1299) cancer cell lines, with IC50 values below 12 mu M, and were also fairly selective when tested in nonmalignant cells. Selenocyanate compounds 1-2a displayed potent antileishmanial activity in L. major and L. infantum, with IC50 values below 5 mu M. They also exhibited antibacterial activity in six bacterial strains, notably in S. epidermidis with MIC and MBC values of 12.5 mu g/mL. Ferrocene-containing selenide 2c was also identified as a potent antileishmanial agent with radical scavenging activity. Remarkably, derivative 2a with a selenocyanate moiety was found to act as a multitarget compound with antiproliferative, leishmanicidal, and antibacterial activities. Thus, the current work showed that 2a could be an appealing scaffold to design potential therapeutic drugs for multiple pathologies.

Mobile genetic elements play an important role in the dissemination of antibiotic resistant bacteria among human and environmental sources. Therefore, the aim of this study was to determine the occurrence and patterns of integrons and insertion sequences of extended-spectrum -lactamase (ESBL)-producing Escherichia coli isolated from different sources in Navarra, northern Spain. A total of 150 isolates coming from food products, farms and feeds, aquatic environments, and humans (healthy people and hospital inpatients), were analyzed. PCRs were applied for the study of class 1, 2, and 3 integrons (intI1, intI2, and intI3), as well as for the determination of insertion sequences (IS26, ISEcp1, ISCR1, and IS903). Results show the wide presence and dissemination of intI1 (92%), while intI3 was not detected. It is remarkable, the prevalence of intI2 among food isolates, as well as the co-existence of class 1 and class 2 (8% of isolates). The majority of isolates have two or three IS elements, with the most common being IS26 (99.4%). The genetic pattern IS26-ISEcp1 (related with the pathogen clone ST131) was present in the 22% of isolates (including human isolates). In addition, the combination ISEcp1-IS26-IS903-ISCR1 was detected in 11 isolates being, to our knowledge, the first study that describes this genetic complex. Due to the wide variability observed, no relationship was determined among these mobile genetic elements and beta-lactam resistance. More investigations regarding the genetic composition of these elements are needed to understand the role of multiple types of integrons and insertion sequences on the dissemination of antimicrobial resistance genes among different environments.