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Significance: The development of an imaging technique to accurately identify biofilm regions on tissues and in wounds is crucial for the implementation of precise surface-based treatments, leading to better patient outcomes and reduced chances of infection. Aim: The goal of this study was to develop an imaging technique that relies on selective trypan blue (TB) staining of dead cells, necrotic tissues, and bacterial biofilms, to identify biofilm regions on tissues and wounds. Approach: The study explored combinations of ambient multi-colored LED lights to obtain maximum differentiation between stained biofilm regions and the underlying chicken tissue or glass substrate during image acquisition. The TB imaging results were then visually and statistically compared to fluorescence images using a shape similarity measure. Results: The comparisons between the proposed TB staining method and the fluorescence standard used to detect biofilms on tissues and glass substrates showed up to 97 percent similarity, suggesting that the TB staining method is a promising technique for identifying biofilm regions. Conclusions: The TB staining method demonstrates significant potential as an effective imaging technique for the identification of fluorescing and non-fluorescing biofilms on tissues and in wounds. This approach could lead to improved precision in surface-based treatments and better patient outcomes.

The first line therapy for Lyme disease is treatment with doxycycline, amoxicillin, or cefuroxime. In endemic regions, the persistence of symptoms in many patients after completion of antibiotic treatment remains a major healthcare concern. The causative agent of Lyme disease is a spirochete, Borrelia burgdorferi, an extreme auxotroph that cannot exist under free-living conditions and depends upon the tick vector and mammalian hosts to fulfill its nutritional needs. Despite lacking all major biosynthetic pathways, B. burgdorferi uniquely possesses three homologous and functional methylthioadenosine/S-adenosylhomocysteine nucleosidases (MTANs: Bgp, MtnN, and Pfs) involved in methionine and purine salvage, underscoring the critical role these enzymes play in the life cycle of the spirochete. At least one MTAN, Bgp, is exceptional in its presence on the surface of Lyme spirochetes and its dual functionality in nutrient salvage and glycosaminoglycan binding involved in host-cell adherence. Thus, MTANs offer highly promising targets for discovery of new antimicrobials. Here we report on our studies to evaluate five nucleoside analogs for MTAN inhibitory activity, and cytotoxic or cytostatic effects on a bioluminescently engineered strain of B. burgdorferi. All five compounds were either alternate substrates and/or inhibitors of MTAN activity, and reduced B. burgdorferi growth. Two inhibitors: 5′-deoxy-5′-iodoadenosine (IADO) and 5′-deoxy-5′-ethyl-immucillin A (dEt-ImmA) showed bactericidal activity. Thus, these inhibitors exhibit high promise and form the foundation for development of novel and effective antimicrobials to treat Lyme disease.

West Nile virus (WNV) causes potentially fatal neuroinvasive disease and persists at endemic levels in many parts of the world. Despite advances in our understanding of WNV pathogenesis, there remains a significant need for a human vaccine. The domain III (DIII) region of the WNV envelope protein contains epitopes that are the target of neutralizing antibodies. We have constructed a chimeric fusion of the non-toxic cholera toxin (CT) CTA2/B domains to DIII for investigation as a novel mucosally-delivered WNV vaccine. Purification and assembly of the chimera, as well as receptor-binding and antigen delivery, were verified by western blot, GM1 ELISA and confocal microscopy. Groups of BALB/c mice were immunized intranasally with DIII-CTA2/B, DIII, DIII mixed with CTA2/B, or CTA2/B control, and boosted at 10 days. Analysis of serum IgG after 14 and 45 days revealed that mucosal immunization with DIII-CTA2/B induced significant DIII-specific humoral immunity and drove isotype switching to IgG2a. The DIII-CTA2/B chimera also induced antigen-specific IgM and IgA responses. Bactericidal assays indicate that the DIII-CTA2/B immunized mice produced DIII-specific antibodies that can trigger complement-mediated killing. A dose escalation resulted in increased DIII-specific serum IgG titers on day 45. DIII antigen alone, in the absence of adjuvant, also induced significant systemic responses after intranasal delivery. Our results indicate that the DIII-CTA2/B chimera is immunogenic after intranasal delivery and merits further investigation as a novel WNV vaccine candidate.