Reply To: Dr. Eva Sapi – Geneticist, Microbiologist (Two Videos Available)

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    Novel Fugitive Strategy for Borrelia burgdorferi: Biofilm
    Luecke David Francis BS, Datar Ak****a BS, Kaur Navroop MS, Bien-Aime H Lubraine BS, Bastian Scott BS, Sinha Saion PhD, Sapi Eva PhD

    Lyme and Tick-borne Diseases Research Group, Department of Biology and Environmental Sciences, University of New Haven, West Haven, CT 06516 USA

    Although antibiotic compounds exist that are demonstrably effective against planktonic Borrelia burgdorferi, the causative agent of Lyme disease, many patients that have been diagnosed with Lyme disease continue to have persistent symptoms that do not respond to treatment. B. burgdorferi is a known pleomorphic species, able to adopt alternative, defensive morphologies to increase antibiotic resistance of the individual. One of these morphologies is the cyst form, which is resistant to the front line antibiotic treatment. Additional compounds are effective against the cyst form, yet still patient symptoms persist.

    Here we have employed several modes of microscopy to characterize another alternative morphology, the biofilm. Among optical microscopy techniques, dark field microscopy was used to observe the interaction of peripheral spirochetes with the biofilm, DIC microscopy revealed the heterogeneity of the biofilm matrix, and fluorescence microscopy enabled observation of the sessile internal biofilm population in a GFP-expressing population. A relatively new technique, atomic force microscopy, was used to directly scan the topography of the biofilm. The ability of B. burgdorferi to assume a biofilmic morphology may partly explain the continuing presence of symptoms in chronic Lyme sufferers. The B. burgdorferi biofilm likely provides a refuge for chronic Lyme infection, and offers an additional avenue of attack for potential treatments for Lyme disease.

    Expression Profile of Quorum Sensing Biomarkers during Biofilm Development in Borrelia burgdorferi
    Bien-Aim H. Lubraine BS, Kaur Navroop MS, Datar Ak****a BS, Luecke David BS, Mpoy Cedric BS, Pabbati Namrataben MS, Sapi Eva Ph.D.

    Lyme and Tick-borne Diseases Research Group, Department of Biology and Environmental Sciences, University of New Haven, West Haven, CT 06516 USA

    We have recently suggested that Borrelia burgdorferi is capable of hiding in a self-made protective layer called biofilm. The main purpose of the biofilm structure is to allow microbes to survive various environmental stresses, including the presence of attacking immune cells or antibacterial agents. While conventional antibiotic therapy is usually effective against free-floating bacteria, it is frequently ineffective once pathogens have formed biofilms, because biofilm colonies can be up to 1,000-times more resistant to antibiotics. To be able to prevent and destroy Borrelia burgdorferi biofilm, we need to better understand the molecular mechanism taking place during biofilm development.

    In this project, we have chosen to monitor specific genetic markers, which regulate communication in the biofilm, so called “quorum sensing”. The quorum sensing biomarkers pfs and luxS are part of the Autoinducer-2 (AI-2) biosynthetic pathway. The pfs enzyme detoxifies S-adenosylhomocysteine (SAH) to S-ribosylhomocysteine (SRH), and the luxS enzyme catalyzes the conversion of SRH to (S)-4,5-dihydroxy-2,3-pentanedione (DPD) and homocysteine. The expression profile of pfs and luxS biomarkers were compared under planktonic and biofilm environments. Borrelia total RNA was extracted in 24-hours increments from 0-hours (planktonic) to 96-hours of biofilm formation, cDNAs were prepared, and Real-time PCR was performed.

    Our results showed that both markers were tightly regulated between the 48 to 96 hours period. LuxS was down regulated from 48 to 72-hours, and up regulated from 72 to 96 hours. Interestingly, pfs yielded to a similar regulation pattern. In summary, our results show that regulation of quorum sensing genes, such as luxS and pfs, is dynamic and provide excellent markers to monitor biofilm development in Borrelia burgdorferi.