List of Abstracts:
- An in vitro evaluation of antibiotic susceptibility of different morphological forms of Borrelia burgdorferi
- Novel Fugitive Strategy for Borrelia burgdorferi: Biofilm
- Expression Profile of Quorum Sensing Biomarkers during Biofilm Development in Borrelia burgdorferi
- Novel Filarial Nematode Infection in Ixodus scapularis Ticks Collected from Southern Connecticut
- Doxycycline Treatment Induces Cyst Formation In Borrelia Burgdorferi
- Survey of Borrelia burgdorferi infection rate in Ixodes scapularis ticks collected in Southern Connecticut in 2007-2008
An in vitro evaluation of antibiotic susceptibility of different morphological forms of Borrelia burgdorferi
Kaur Navroop MS, Datar Ak****a BS, Luecke David BS, Bien-Aim H. Lubraine BS, Mpoy Cedric BS, Pabbati Namrata 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
A tick borne, multisystemic disease, Lyme borreliosis caused by the spirochete Borrelia burgdorferi has grown into a major public health problem during last ten years. The primary treatment for chronic Lyme disease is administration of various antibiotics. However, relapse of the disease often occurs when antibiotic treatment is discontinued. It is suggested that this resistance and reoccurrence of Lyme disease might be due to formation of different morphological forms of Borrelia burgdorferi. The two major known resistant morphologies are cyst and biofilm, which forms in response to stress conditions such as exposure to antibiotics.
To be able to provide novel and effective therapeutic approaches for physicians to explore the treatment options for chronically ill Lyme disease patients, we need to better understand the direct effect of antibiotics on the different morphological forms of Borrelia burgdorferi. In this study, we tested an in vitro susceptibility of several morphological forms of Borrelia burgdorferi to different antibacterial agents such as tetracyclines, hydroxycholoroquine and 5- nitroimidazoles. Cell viability assays have been performed before and after the administration of the different drugs to cultures of Borrelia burgdorferi and different microscopic techniques such as dark field and fluorescent have been used to monitor those morphological forms of Borrelia burgdorferi.
Our study suggested that exposure of Borrelia burgdorferi cultures to concentrations greater than minimum bactericidal concentration (MBC) of doxycycline (>25µg/ml) and hydroxychloroquine (Plaquenil) (>50µg/ml) significantly reduced the spirochete population but unfortunately also increased the number of cystic forms. However, similar treatment of 5- nitroimidazoles such as metronidazole (greater than MBC as >35µg/ml) and tinidazole (greater than MBC as >32µg/ml) led to reduction of cystic forms in the culture. Furthermore, when combinations of most effective concentrations of 5- nitroimidazoles and tetracycline were tested in vitro, both cystic and spirochete forms of Borrelia burgdorferi were significantly eliminated Our study suggests that Borrelia burgdorferi specific combination therapy for Lyme disease patients might provide treatment option with a better outcome.
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.
Novel Filarial Nematode Infection in Ixodus scapularis Ticks Collected from Southern Connecticut
Pabbati Namrata M.S., Madari Shilpa B.S., Reddy Raghvendar B.S., Kaur Navroop M.S., Datar Ak****a B.S., Patel Seema B.S., Luecke David B.S., Bien-Aim H. Lubraine B.S., Mpoy Cedric B.S., Rossi Michael Ph.D., 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.
Tick-borne co-infections in Lyme disease patients are of mounting concern because they have been found to increase the severity and duration of acute illness. In the last decade, there were a numbers of efforts to identify potential co-infection in Lyme disease patients, with the goal of providing a rational for more specific treatments. Most investigators focused on identifying novel tick- borne bacteria, viruses and fungal infection other than B. burgdoferi in ticks or in patients with a tick bite history.
Despite all these efforts, there were no improvements in some patients with tick bite history even after they introduced novel treatment protocols. This observation raises an important question as to whether species other than bacteria, virus or fungus could responsible for these chronic problems. Dr. Willy Burgdorfer found 30 microfilarial worms (species was not identified) in one adult Ixodis dammini tick in Shelter Island NY in mid 80s. Based on this single study, we hypothesized that the Ixodes scapularis (deer tick) is a potential vector for filarial parasites and to prove that hypotheses we used cell and molecular biology techniques such as polymerase chain reaction, directs sequencing and in situ hybridization methods.
First, we amplified filarial nematode specific sequences using different filarial nematode genus specific primers such as FL1, FL2, 12s rRNA, ITS (internal transcribed sequences). These PCR results showed four sequences showing high identity with filarial nematode sequences. >From these sequences, Onchocerca species showed the closest nucleotide similarity, therefore Onchocerca species-specific primers were used such as OVMSP2 (O. volvulus major sperm protein), NADH (nicotinamide adenine dinucleotide dehydrogenase) and 16s rRNA to confirm the exact species. Other techniques such as direct staining of paraffin sections of tick samples and nematode specific in-situ hybridization methods are also utilized to further support the presence of live filarial worms in deer ticks and revealed that larval filarial nematode indeed harbor the gut tissue of deer ticks. The significance to this co-infection is that if it is in fact tick-borne and transmittable to humans, it could explain why extensive antibiotic treatment for patients with a tick-bite history could fail. The result from these studies could provide a novel therapeutic target for our physicians to explore for those chronically ill patients with tick bite history.
Doxycycline Treatment Induces Cyst Formation In Borrelia Burgdorferi
Datar Ak****a B.S., Kaur Navroop M.S., Luecke David B.S., Bien-Aim H. Lubraine B.S., Mpoy Cedric B.S., Pabbati Namrata M.S., 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
Lyme disease is caused by infection with the spirochete Borrelia burgdorferi and is transmitted to humans by tick bites of the genus Ixodes. The primary treatment for Lyme disease is tetracyclines such as doxycycline. However, recent clinical studies show very little improvement rate for Lyme disease patients after using doxycycline, even for years. One possible explanation for this observation is that B. burgdorferi can change from their vegetative spirochete form into a cystic form after exposure to antibiotics. In this study, we tested effectiveness of doxycycline on the various morphological forms of B. burgdorferi in vitro at different concentrations (concentrations > 25 µg/ml, which is the minimum bactericidal concentration of doxycycline).
To determine the effectiveness of doxycycline, cell proliferative assay and microscopic techniques such as dark field microscopy and fluorescent microscopy were utilized. For Borrelia culture we used a culture which expresses a green fluorescent protein, to help to monitor the effect of doxycycline treatment on the different morphological forms. Our results showed that 250 µg/ml treatment of doxycycline eliminated 90% of spirochete form of B. burgdorferi, but it also increased the cystic formation very significantly. Our findings provide a probable explanation for persistent symptoms in Lyme disease patients even after years of doxycycline treatment because while doxycycline can eliminate the spirochete form of B. burgdorferi, it also pushes Borrelia spirochetes into an antibiotic resistant cystic form.
Survey of Borrelia burgdorferi infection rate in Ixodes scapularis ticks collected in Southern Connecticut in 2007-2008
1Cedric Mpoy B.S, 1Datar Ak****a BS, 1Bien-Aim H. Lubraine BS, 1Pabbati Namrata MS, Georgina Scholl M.D, Ph.D, and 1Eva Sapi Ph.D.
1Lyme and Tick-borne Diseases Research Group, Department of Biology and Environmental Sciences, University of New Haven, West Haven, CT 06516 USA, 2 Fairfield County Municipal Deer Management Alliance, Georgetown, CT 06829
In the Fall of 2007, the University of New Haven begun one of the largest tick collection and Borrelia burgdorferi testing surveys in US; testing over 50 sites in Fairfield County CT. The uniqueness of this survey is that the tick collection is being performed at school yards, public parks and playgrounds, The collection sites were designed to represent sites where children or families have outside activity to evaluate the risk of exposure of our children to tick borne diseases. Ixodes scapularis (deer ticks) were collected for ~90 minutes, within a few yard perimeters from these playgrounds at each site, by at least 4 people using the dragging method. The live deer ticks were transported to the University of New Haven where further analyzed for the presence of Ixodes scapularis (control) and Borrelia burgdorferi DNAs using the standard and real time polymerase chain reaction as well as direct sequencing methods.
As of October 2009, the University of New Haven has collected ~2000 deer tick samples and tested ~1500 samples for Borrelia burgdorferi (Bb) infection. The overall Bb infection rate was 72%, ranging from 51%-94% in 2007 and 90%, ranging from 88%-96%. Our result strongly suggests that there is a very significant exposure risk for tick-borne infections around school yards and playgrounds in Fairfield County CT.
