Engineering Cyborg bacteria as vaccines against antibiotic-resistant bacteria

Introduction:: Death rates due to antibiotic-resistant microbial infection are increasing, and the approval of new antibiotic classes is falling. Several types of solutions are being pursued, ranging from new antibiotics to vaccines. Vaccines are attractive solutions because they can provide long-lasting immunity against the deadly pathogen, which can reduce the need for antibiotics and, in turn, help to slow the development of antibiotic resistance. However, existing vaccines based on bacterial cell-wall polysaccharides, killed bacteria, or bacterial toxins are poorly immunogenic. Live-attenuated bacterial vaccines carry the risk of outgrowth and are unsafe for immunocompromised patients. As a result, there are no approved vaccines for the most prevalent bacterial pathogens. An urgent need is to develop potent and safe vaccines against antibiotic-resistant pathogens, specifically methicillin-resistant Staphylococcus aureus. Here, we create non-replicating-but-metabolic-active Cyborg Bacteria that can be used as a potential vaccine candidate for S. aureus infection (Fig. A). Cyborg Bacteria are generated by hydrogel crosslinking inside the bacterial cytoplasm [1]. The Cyborg Bacteria preserve the genetic material integrity, fluid and functional cell membrane interfaces, and active metabolic pathways. The Cyborg Bacteria are investigated using molecular, in vitro, and in vivo experiments to study their ability to induce both humoral and cell-mediated immune responses. Furthermore, due to their non-replicative ability, the Cyborg Bacteria have reduced risks of causing undesired infections or other side effects.

Materials and Methods:: To create Cyborg Bacteria, bacteria were incubated in hydrogel buffer composed of polyethylene glycol diacrylate, photoactivator, and fluorescein diacrylate. The hydrogel buffer was incorporated inside the bacteria through freeze-thaw cycles. After thawing and recovery in fresh media, the hydrogel inside the bacteria was crosslinked using UV irradiation [1]. The Cyborg Bacteria were then washed with PBS and resuspended in appropriate media. For in vitro studies gentamycin protection assay was performed with RAW 264.7 macrophages cell lines. Both Cyborg and wildtype S. aureus were allowed to infect the macrophages with multiplication of infection 1:50. Bacterial colocalization in macrophages was studied using confocal microscopy. Furthermore, absolute quantification bacterial load inside the macrophages was estimated by qPCR of the 16s rRNA gene in infected cells. The DNA concentration of 0.1 OD culture was noted, and 10-fold dilutions were used to construct the standard curves for 16s rRNA gene amplification. Bacterial load of wild type and Cyborg Bacteria was estimated by plotting their Ct values in a standard curve. Next, the effectiveness of Cyborg Bacteria as a vaccine in preventing or reducing the severity of infection was studied by a wound assay in mice. Parameters like wound size, inflammation, and tissue repair at regular intervals were recorded to study the efficacy of Cyborg S. aureus as a vaccine [2].


Results, Conclusions, and Discussions:: We have created at least 4 different Cyborg bacteria using different strains of methicillin-resistant S. aureus, E. coli, Pseudomonas aeruginosa, and Klebsiella Pneumonia. Flow cytometry analysis (Fig. B) and colony counts show that these Cyborg Bacteria maintain high metabolic activity and a nonreplicating phenotype. Flow cytometry analysis of Cyborg Bacteria shows a similar shift as wild type when stained with CTC (5-cyano-2,3-ditolyl tetrazolium chloride) dye, which reports the respiratory activity of bacteria. The results suggest that they have similar respiratory activity. As expected, due to their non-replicating property, Cyborg Bacteria show negligible colony-forming-unit compared to the wild type. Transmission electron microscopy shows the cytoplasmic changes in Cyborg Bacteria due to intracellular hydrogelation (Fig. C). In vitro studies indicate the equivalent uptake of Cyborg Bacteria by macrophages as the unmodified bacteria (Fig. D). Specifically, confocal microscopy images show similar colocalization of both wild type and Cyborg Bacteria (Green) inside the macrophages stained with phalloidin (Red). The results show that hydrogelation of bacteria does not significantly alter their ability to interact with host immune cells. Quantitative PCR shows that both Cyborg and wild type bacteria show similar invasion rates into macrophages (Fig. E). The Cyborg Bacteria are then investigated for their vaccine efficacy in a C57BL/6 mouse infection model.



We have generated Cyborg Bacteria using both Gram-positive and Gram-negative bacterial pathogens. We have validated the ability of Cyborg bacteria to invade macrophages. The Cyborg Bacteria are then compared to existing vaccine approaches in generating immune responses. Overall, the results show that Cyborg S. aureus is a promising vaccine candidate against MRSA infections.


Acknowledgements (Optional): : We thank NIGMS for supporting the work. We also thank the advice and support of all members of the Tan lab.


References (Optional): : < !1. Contreras‐Llano LE, Liu YH, Henson T, Meyer CC, Baghdasaryan O, Khan S, Lin CL, Wang A, Hu CM, Tan C. Engineering Cyborg Bacteria Through Intracellular Hydrogelation. Advanced Science. 2023 Jan 11:2204175.
< ! 2. Cho JS, Guo Y, Ramos RI, Hebroni F, Plaisier SB, Xuan C, Granick JL, Matsushima H, Takashima A, Iwakura Y, Cheung AL. Neutrophil-derived IL-1β is sufficient for abscess formation in immunity against Staphylococcus aureus in mice. PLoS pathogens. 2012 Nov 29;8(11):e1003047