https://www.youtube.com/watch?v=qXTPiJWhtDg
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Northeastern University Antimicrobial Discovery Center
Kim Lewis, "The Paradox of Chronic Infections"
All bacterial populations stochastically produce a small number of dormant persister cells tolerant to antibiotics. Persisters are not mutants but phenotypic variants of the wild type.
We are interested in discovering the mechanism of persister formation. Using cell sorting and transcriptome analysis, we find that chromosomally-encoded “toxin” genes act to shut down cellular functions, creating a dormant state. Examples include HipA, which encodes a protein kinase that phosphorylates elongation factor Ef-Tu, blocking protein synthesis, and TisB, a small peptide that inserts in the membrane, causing a drop in pmf and ATP levels. Interestingly, TisB synthesis is induced by DNA damaging agents, including fluoroquinolone antibiotics. This means that persisters can be formed not only stochastically, but through stress response mechanisms.
We find that in chronic infections such as cystic fibrosis, antibiotic treatment selects for high-persister mutants. Whole genome sequencing indicates the mechanism for increased production of persisters.
Current projects involve the study of the molecular mechanisms of persister formation governed by HipA and TisB in E. coli; the search for persister genes in P. aeruginosa, S. aureus and M. tuberculosis; and characterization of high-persister mutants from clinical isolates of these pathogens. A related project is discovery of compounds capable of eliminating persisters. The work of the persister group is supported by a Transformative Award from the NIH, and with grants from ARO and CF Foundation.
We are interested in discovering the mechanism of persister formation. Using cell sorting and transcriptome analysis, we find that chromosomally-encoded “toxin” genes act to shut down cellular functions, creating a dormant state. Examples include HipA, which encodes a protein kinase that phosphorylates elongation factor Ef-Tu, blocking protein synthesis, and TisB, a small peptide that inserts in the membrane, causing a drop in pmf and ATP levels. Interestingly, TisB synthesis is induced by DNA damaging agents, including fluoroquinolone antibiotics. This means that persisters can be formed not only stochastically, but through stress response mechanisms.
We find that in chronic infections such as cystic fibrosis, antibiotic treatment selects for high-persister mutants. Whole genome sequencing indicates the mechanism for increased production of persisters.
Current projects involve the study of the molecular mechanisms of persister formation governed by HipA and TisB in E. coli; the search for persister genes in P. aeruginosa, S. aureus and M. tuberculosis; and characterization of high-persister mutants from clinical isolates of these pathogens. A related project is discovery of compounds capable of eliminating persisters. The work of the persister group is supported by a Transformative Award from the NIH, and with grants from ARO and CF Foundation.
Lyme disease, caused by the spirochete Borrelia burgdorferi, is the most common vector-borne infection in North America and Europe. In 10-20% of cases, patients develop chronic Lyme disease after completing antibiotic treatment. The cause of these chronic symptoms is, however, poorly understood. We have previously shown that high-persister mutants are selected for over the course of relapsing chronic infections of Pseudomonas aeruginosa in cystic fibrosis patients and Candida albicans in oral thrush patients. It seems likely that these high persister mutants may contribute to the recalcitrance of the infection. Persister cells are drug-tolerant phenotypic variants of normal cells and may cause recurrent bacterial infections by resuming growth once antibiotic treatment has ceased. We hypothesize that persister cells play a role in the treatment failure that leads to chronic Lyme disease. Here, using time-dependent and dose dependent survival assays, we show that B. burgdorferi forms persister cells to the antibiotics commonly used for treatment of Lyme disease. Our results indicate that in a B.burgdorferi population, 0.001% to 1% of the cells can survive lethal doses of various antibiotics in vitro. These persister cells may contribute to treatment failure in chronic Lyme patients. Future experiments are aimed at screening for a better antimicrobial therapy to eradicate persisters in B. burgdorferi.
http://www.abstractsonline.com/Plan/ViewAbstract.aspx?mID=3475&sKey=ddd2449f-2f20-40f0-b4e3-23d73fdf5fae&cKey=6420d060-6ac4-4246-b649-bf393a7997df&mKey=673511f0-c86b-432f-a387-058032b8500b
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Borrelia burgdorferi, the causative agent of Lyme disease, forms drug-tolerant persister cells.
Bijaya Sharma, Autumn V. Brown, Nicole E. Matluck, Linden T. Hu and Kim Lewis
In this study, we examined the ability of B. burgdorferi to form persisters. Killing of growing cultures of B. burgdorferi with antibiotics used to treat the disease was distinctly biphasic, with a small subpopulation of surviving cells. Upon regrowth, these cells formed a new subpopulation of antibiotic-tolerant cells, indicating that these are persisters rather than resistant mutants. The level of persisters increased sharply as the culture transitioned from exponential to stationary phase. Combinations of antibiotics did not improve killing. Daptomycin, a membrane-active bactericidal antibiotic, killed stationary phase cells, but not persisters. Mitomycin C, an anti-cancer agent that forms adducts with DNA, killed persisters and eradicated both growing and stationary cultures of B. burgdorferi. Finally, we examined the ability of pulse-dosing an antibiotic to eliminate persisters. After addition of ceftriaxone, the antibiotic was washed away, surviving persisters were allowed to resuscitate, and antibiotic was added again. Four pulse-doses of ceftriaxone killed persisters, eradicating all live bacteria in the culture.
http://aac.asm.org/content/early/2015/05/20/AAC.00864-15
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What everyone was waiting for – Possible Lyme Disease Cure on Horizon
Gepubliceerd op 13 nov. 2014
All the lies are starting to come out folks, breaking news has just surfaced that Ying Zhang, MD, PhD developed a test that can identify persister cells in Chronic Lyme Disease. Borrelia burgdorferi persister cells neither die nor grow in the presence of an antibiotic. Rather, they exist in a dormant state that allows them to survive antibiotic treatment, only to awaken later and start a new wave of infection. This new test will be able to quantify how many Borrelia burgdorferi are alive and how many are dead after each drug was added to the bacteria. The method stains the living bacteria green and the dead or dying bacteria red in a way that filters out the noise that can corrupt existing tests. So now, it's only a matter of time till the CDC changes their protocol and week can seek immediate antibitiotic treatment.
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EFFECTIVENESS OF STEVIA REBAUDIANA WHOLE LEAF EXTRACT AGAINST THE VARIOUS MORPHOLOGICAL FORMS OF BORRELIA BURGDORFERI IN VITRO
P. A. S. Theophilus, M. J. Victoria, K. M. Socarras, K. R. Filush, K. Gupta, D. F. Luecke, E. Sapi*
Department of Biology and Environmental Science, University of New Haven, West Haven, CT, USA
Received: September 7, 2015; Accepted: October 26, 2015 | European Journal of Microbiology and Immunology (2015) | DOI: 10.1556/1886.2015.00031
Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treatment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains unknown, but recent studies suggested the possibilities of the presence of antibiotic resistant Borrelia persister cells and biofilms.
In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferi spirochetes, persisters, and biofilm forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to different microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combinations. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Subculture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics were tested against attached biofilms, Stevia significantly reduced B. burgdorferi forms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi.
P. A. S. Theophilus, M. J. Victoria, K. M. Socarras, K. R. Filush, K. Gupta, D. F. Luecke, E. Sapi*
Department of Biology and Environmental Science, University of New Haven, West Haven, CT, USA
Received: September 7, 2015; Accepted: October 26, 2015 | European Journal of Microbiology and Immunology (2015) | DOI: 10.1556/1886.2015.00031
Lyme disease is a tick-borne multisystemic disease caused by Borrelia burgdorferi. Administering antibiotics is the primary treatment for this disease; however, relapse often occurs when antibiotic treatment is discontinued. The reason for relapse remains unknown, but recent studies suggested the possibilities of the presence of antibiotic resistant Borrelia persister cells and biofilms.
In this study, we evaluated the effectiveness of whole leaf Stevia extract against B. burgdorferi spirochetes, persisters, and biofilm forms in vitro. The susceptibility of the different forms was evaluated by various quantitative techniques in addition to different microscopy methods. The effectiveness of Stevia was compared to doxycycline, cefoperazone, daptomycin, and their combinations. Our results demonstrated that Stevia had significant effect in eliminating B. burgdorferi spirochetes and persisters. Subculture experiments with Stevia and antibiotics treated cells were established for 7 and 14 days yielding, no and 10% viable cells, respectively compared to the above-mentioned antibiotics and antibiotic combination. When Stevia and the three antibiotics were tested against attached biofilms, Stevia significantly reduced B. burgdorferi forms. Results from this study suggest that a natural product such as Stevia leaf extract could be considered as an effective agent against B. burgdorferi.
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Peer Reviewed Evidence of Persistence of Lyme Disease Spirochete Borrelia burgdorferi and Tick-Borne Diseases
The following is a list of over 700 peer reviewed articles that support the evidence of persistence of Lyme and other tick-borne diseases. It is organized into different categories—general, psychiatric, dementia, autism and congenital transmission.
http://www.ilads.org/ilads_news/wp-cont ... nce-V2.pdf
The following is a list of over 700 peer reviewed articles that support the evidence of persistence of Lyme and other tick-borne diseases. It is organized into different categories—general, psychiatric, dementia, autism and congenital transmission.
http://www.ilads.org/ilads_news/wp-cont ... nce-V2.pdf
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Researchers investigate four promising new treatments for Lyme disease
March 29, 2016 by Thea Singer
A new regimen
Lewis and his colleagues are providing that focus. A subpopulation of B. burgdorferi cells, they discovered earlier, are “persister” cells—they are alive but lie dormant, in a sporelike state. Because antibiotics attack only actively functioning bacterial cells, persisters escape the onslaught. However, once the antibiotic has been flushed from the system, the persisters “wake up,” says Lewis, dividing and multiplying until an army of progeny infect the host.
That’s where “pulse dosing” comes in. Lewis’ team, in collaboration with researchers studying B. burgdorferi in mice at Tufts University’s School of Medicine, has been analyzing the effect of giving the mice an antibiotic that kills all the actively functioning bacterial cells and then—using the timing that eradicated the pathogen in the test tube—giving additional doses to quash the persister cells as they begin to wake up but before they reproduce.
Plans are in the works for the first pulse-dosing human trials with medical schools.
Drugs combined and discovered
Doxycycline may be standard first-line treatment for Lyme, but, says Lewis, it doesn’t even kill B. burgdorferi, it just suppresses its growth, leaving the rest of the work to the immune system. “We simply asked the question: ‘Is it possible to combine existing antibiotics to treat not only chronic Lyme but any stage of Lyme if the diagnosis is unambiguous?”
The researchers have already found combinations that are effective against the B. burgdorferi in the test tube and will move on to animal studies next.
They are tackling new-drug discovery on two fronts: Plumbing the 200,000-plus compounds in their collection at Northeastern to find the ones that act solely against B. burgdorferi to avoid unwanted side effects and, in collaboration with Novobiotic Pharmaceuticals, extracting drugs from bacteria that live in soil using the iChip, a device developed by Slava Epstein, Distinguished Professor at Northeastern, in collaboration with Lewis. The iChip provides access to the 99 percent of microbes in the environment that heretofore could not be grown in the lab.
“So far we have identified two lead compounds that kill B. burgdorferi and have no activity against other bacteria,” says Lewis.
The researchers are also exploring whether the microbiome has “shifted” in those with PTLDS, to see whether introducing certain microorganisms might shift it back. Animal studies have shown that manipulating the microbiome composition alleviates symptoms of autoimmune diseases such as rheumatoid arthritis, which share many characteristics with PTLDS.
“We are going at Lyme disease with everything we have,” says Lewis.
March 29, 2016 by Thea Singer
A new regimen
Lewis and his colleagues are providing that focus. A subpopulation of B. burgdorferi cells, they discovered earlier, are “persister” cells—they are alive but lie dormant, in a sporelike state. Because antibiotics attack only actively functioning bacterial cells, persisters escape the onslaught. However, once the antibiotic has been flushed from the system, the persisters “wake up,” says Lewis, dividing and multiplying until an army of progeny infect the host.
That’s where “pulse dosing” comes in. Lewis’ team, in collaboration with researchers studying B. burgdorferi in mice at Tufts University’s School of Medicine, has been analyzing the effect of giving the mice an antibiotic that kills all the actively functioning bacterial cells and then—using the timing that eradicated the pathogen in the test tube—giving additional doses to quash the persister cells as they begin to wake up but before they reproduce.
Plans are in the works for the first pulse-dosing human trials with medical schools.
Drugs combined and discovered
Doxycycline may be standard first-line treatment for Lyme, but, says Lewis, it doesn’t even kill B. burgdorferi, it just suppresses its growth, leaving the rest of the work to the immune system. “We simply asked the question: ‘Is it possible to combine existing antibiotics to treat not only chronic Lyme but any stage of Lyme if the diagnosis is unambiguous?”
The researchers have already found combinations that are effective against the B. burgdorferi in the test tube and will move on to animal studies next.
They are tackling new-drug discovery on two fronts: Plumbing the 200,000-plus compounds in their collection at Northeastern to find the ones that act solely against B. burgdorferi to avoid unwanted side effects and, in collaboration with Novobiotic Pharmaceuticals, extracting drugs from bacteria that live in soil using the iChip, a device developed by Slava Epstein, Distinguished Professor at Northeastern, in collaboration with Lewis. The iChip provides access to the 99 percent of microbes in the environment that heretofore could not be grown in the lab.
“So far we have identified two lead compounds that kill B. burgdorferi and have no activity against other bacteria,” says Lewis.
The researchers are also exploring whether the microbiome has “shifted” in those with PTLDS, to see whether introducing certain microorganisms might shift it back. Animal studies have shown that manipulating the microbiome composition alleviates symptoms of autoimmune diseases such as rheumatoid arthritis, which share many characteristics with PTLDS.
“We are going at Lyme disease with everything we have,” says Lewis.
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WATCH: Bacteria Invade Antibiotics And Transform Into SuperbugsSeptember 8, 2016
If you've ever wanted to watch a superbug evolve before your very eyes, you're in luck. Researchers filmed an experiment that created bacteria a thousand times more drug-resistant than their ancestors. In the time-lapse video, a white bacterial colony creeps across an enormous black petri dish plated with vertical bands of successively higher doses of antibiotic.
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And if scientists can see it, maybe they can start to study it. Using something as simple as a giant petri dish like this could help scientists open up that spatial dimension that has been missing from the lab, says Pamela Yeh, a microbiologist at UCLA who was not involved in the experiment. "Hopefully this will put back in people's minds how important the spatial element can be."
If you've ever wanted to watch a superbug evolve before your very eyes, you're in luck. Researchers filmed an experiment that created bacteria a thousand times more drug-resistant than their ancestors. In the time-lapse video, a white bacterial colony creeps across an enormous black petri dish plated with vertical bands of successively higher doses of antibiotic.
..
And if scientists can see it, maybe they can start to study it. Using something as simple as a giant petri dish like this could help scientists open up that spatial dimension that has been missing from the lab, says Pamela Yeh, a microbiologist at UCLA who was not involved in the experiment. "Hopefully this will put back in people's minds how important the spatial element can be."
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A. baumannii is evolving rapidly.........
Erythromycin was an early example; introduced as an alternative to penicillin for the treatment of S. aureus in Boston City Hospital in the early 1950s, it was completely withdrawn after less than a year because 70% of all the S. aureus isolates were found to have become erythromycin resistant. The same was observed with chlortetracycline and chloramphenicol and, subsequently, with other antibiotics
Erythromycin was an early example; introduced as an alternative to penicillin for the treatment of S. aureus in Boston City Hospital in the early 1950s, it was completely withdrawn after less than a year because 70% of all the S. aureus isolates were found to have become erythromycin resistant. The same was observed with chlortetracycline and chloramphenicol and, subsequently, with other antibiotics
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