Uncovering the hidden bacterial ghost communities of yeast and experimental evidences demonstrates yeast as thriving hub for bacteria

Our major concern was to address “yeast endobacteria” which was based on a few reports in the recent past where bacteria may find yeast as a niche for survival. In this study, we report the microbiota of twenty-nine axenic yeast cultures recovered from different habitats based on their 16S rRNA gene-amplicon metagenomes. Yeasts were identified based on D1/D2 or ITS gene sequences. Bacterial diversity was widespread, varied and rich among all yeasts except for four strains. Taxa belonging to the phylum Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes and the genera; Streptococcus, Propionibacterium were common to all the yeasts. Candida tropicalis was used as a model organism to confirm bacteria through fluorescence in situ hybridization (FISH), isolating and re-introducing the isolated bacteria into the yeast. FISH analysis confirmed the endobacteria of C. tropicalis and we have successfully isolated four bacteria only after lysis and disruption of yeast cells. These bacteria were identified as species of Pseudomonas, Chryseobacterium, Lysinibacillus and Propionibacterium. Guestimates indicate 95% of bacterial species of C. tropicalis are yet-to-be-cultivated. We have successfully reintroduced mCherry tagged Pseudomonas into C. tropicalis. Also, auto-fluorescent Prochlorococcus and Rhodopseudomonas could be introduced into C. tropicalis while mCherry tagged E. coli or Salmonella could not be introduced. FISH analysis confirmed the presence of both native and infected bacterial cells present in C. tropicalis. Our findings unveil the insights into the ghost microbiota associated with yeast, which otherwise are considered to be axenic cultures. Their inherent occurrence, together with co-cultivation experiments under laboratory conditions suggests that yeasts are a thriving hub for bacterial communities.

Design, development, and evaluation of the efficacy of a nucleic acid-free version of a bacterial ghost candidate vaccine against avian pathogenic E. coli (APEC) O78:K80 serotype

One of the major bacterial infectious diseases in the poultry industry is avian pathogenic Escherichia coli (APEC), which causes colibacillosis in chickens. To develop a novel nucleic acid-free bacterial ghost (BG) vaccine against the O78:K80 serotype of APEC, in this study we constructed a plasmid that harbored E-lysis and S nuclease (SNUC). Following the expression, the O78:K80 bacteria lost all of their cytoplasmic content and nucleic acids by enzymatic digestion. The functionality of these two proteins in the production procedure of bacterial ghosts was confirmed by monitoring the number of colonies, scanning electron microscopy imaging, gel electrophoresis of genomic DNA, and qPCR on the plasmid content of bacterial ghosts. The protective efficacy of the ghost vaccine generated from O78:K80 serotype of APEC was tested in chickens by injection and inhalation routes and compared with that in chickens that received the injection of a killed vaccine. The O78:K80 BG vaccine candidate, used as injection and inhalation, in comparison with the killed vaccine, triggered higher proinflammatory cytokine expression including IL-6, IL-1β, and TNFSF15; a higher level of antibody-dependent humoral (IgY and IgA) and cellular immune responses (IFNγ and lymphocyte proliferation); and lower lesion scores. According to the results of this study, we suggest that the bacterial ghost technology has the potential to be applied for the development of novel vaccines against avian colibacillosis. This technology provides an effective and reliable approach to make multivalent vaccines for more prevalent APEC strains involved in the establishment of this infectious disease in the poultry industry.

Study of chemical-based induced bacterial ghost applied in vaccine production

Introduction: Bacterial ghosts (BGs), known as the empty cell envelope of gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts, are widely studied and used as the platform for the production of the vaccines as well as the transporting drug and gene delivery. However, the study related to the creation of BGs based on gene expression is still limited because of the difference in cell wall structure between microorganisms. Therefore, in the current study, for the aims to determine chemicals combination and minimum inhibition concentration (MIC) to optimize BGs production. Material and method: Salmonella choleraesuis strain was collected from NAVETCO company. The study used critical concentrations from chemical combination to convert salmonella cells to BGs. Chemicals combination and MIC, temperature, shaking speed were optimized using Plakett- Burman matrix and response surface methodology. Cell structure was determined by using a scanning electron microscope, experimental mice were vaccinated and challenged with virulence to determine immune responses of bacterial ghost. Results: The appropriate chemicals for the production of BGs biomass were NaOH 3.125 mg/ml; SDS 1.15 mg/ml, H2O2 8.79 µl/ml, ethanol. The observation of morphology, BGs have remained the structure and shape, which were like the living microbial cells. Conclusions: The conditions of BGs production have been identified to produce large amounts of bacterial ghost biomass to further application in vaccine production and pharma.