Display of Heterologous Proteins in Bacillus Subtilis Biofilms for Enteric Immunization

One of the foremost goals in vaccine development is the design of effective, heat-stable vaccines that simplify the distribution and delivery while conferring high levels of protective immunity. Here, we describe a method for developing a live, oral vaccine that relies on the biofilm-forming properties of the spore-former bacterium Bacillus subtilis. The amyloid protein TasA is an abundant component of the extracellular matrix of the biofilms formed by B. subtilis that can be genetically fused to an antigen of interest. Spores of the recombinant strain are then prepared and applied via the oral route in an animal model. Due to the intrinsic resistance of the spores, they can bypass the stomach barrier, germinate, and subsequently colonize the gut, where they develop into biofilms, expressing the antigen of interest. We describe here the steps necessary to produce spores, immunization, and downstream analysis of the vaccine efficacy. Keywords: Bacillus subtilis; Biofilm; Endospores; Heterologous; Immunization

Animal petting zoos as sources of Shiga toxin-producing Escherichia coli, Salmonella and extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae

Animal petting zoos and farm fairs provide the opportunity for children and adults to interact with animals, but contact with animals carries a risk of exposure to zoonotic pathogens and antimicrobial-resistant bacteria. The aim of this study was to assess the occurrence of Shiga toxin-producing Escherichia coli (STEC), Salmonella, extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureus (MRSA) in animal faeces from six animal petting zoos and one farm fair in Switzerland. Furthermore, hygiene facilities on the venues were evaluated. Of 163 faecal samples, 75 contained stx1, stx2 or stx1/stx2 genes, indicating the presence of STEC. Samples included faeces from sika deer (100%), sheep (92%), goats (88%), mouflons (80%), camels (62%), llamas (50%), yaks (50%), pigs (29%) and donkeys (6%), whereas no stx genes were isolated from faeces of calves, guinea pigs, hens, ostriches, ponies, zebras or zebus. Salmonella enterica subsp. enterica serovar Stourbridge (S. Stourbridge) was detected in faecal samples from camels. A total of four ESBL-producing E. coli strains were isolated from faeces of goats, camels and pigs. PCR and sequencing identified the presence of blaCTX-M-15 in three and blaCTX-M-65 in one E. coli. Antimicrobial resistance profiling using the disk diffusion method revealed two multidrug-resistant (MDR) E. coli with resistance to ciprofloxacin, gentamicin and azithromycin, all of which are critically important drugs for human medicine. Multilocus sequence typing identified E. coli ST162, E. coli ST2179, extraintestinal high-risk E. coli ST410 and E. coli ST4553, which belongs to the emerging extraintestinal clonal complex (CC) 648. No MRSA was detected. On all animal petting venues, there were inadequacies with regard to access to hygiene information and handwashing hygiene facilities. This study provides data that underscore the importance of hygiene measures to minimize the risk of transmission of zoonotic pathogens and MDR, ESBL-producing E. coli to visitors of animal petting venues