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czwartek, 31 marca 2016

persisters - Borelioza.Trudne do wyeliminowania.Formy przetrwalnikowe.

Nie zaobserwowano aby bakteria  B. burgdorferi była odporna na antybiotyki. Problem leczenia choroby w późnym stadium pomimo podawania antybiotykow - związana jest z obecnościa persisters. 

W poniższym badaniu - badano zdolność B. burgdorferi do wytwarzania  persisters. Zabijanie rosnących kultur B. burgdorferi za pomocą antybiotyków stosowanych w leczeniu tej choroby był  dwufazowy.Jedne ginely a te ,które przeżyły  znowu tworzą nową podgrupę komórek odporną na antybiotyki, co wskazuje, że są to persisters . Kombinacje antybiotyków nie zabiły  pozostałych bakterii.. Stosowano : Daptomycyna, Mitomycyna C - nie zginęły wszystkie Persisters

Wreszcie pulsacyjne podawanie  antybiotyku, aby wyeliminować persisters. Po podaniu ceftriaksonu antybiotyk został wymyty,persisters które przeżyły  mogły się uaktywnić .Wtedy ponownie podano antybiotyk. Cztery dawki impulsów ceftriaksonu zabiły persisters.Nastapilo wyeliminowanie wszystkich żywych bakterii z  hodowli.

http://www.ncbi.nlm.nih.gov/pubmed/26014929

Borrelia burgdorferi, the causative agent of Lyme disease, forms drug-tolerant persister cells

RESULTS

Characterization of B. burgdorferi persisters.
The presence of persisters is indicated by a biphasic pattern in a time-dependent killing experiment. The bulk of the population is rapidly killed, followed by a lower rate of death in a subpopulation of tolerant cells (, ). In order to determine whether B. burgdorferi forms persisters, time-dependent killing experiments were performed with antibiotics commonly prescribed to patients with Lyme disease. Doxycycline is a bacteriostatic protein synthesis inhibitor; amoxicillin and ceftriaxone inhibit bacterial cell wall synthesis and are bactericidal for many bacteria. MICs of doxycycline, amoxicillin, and ceftriaxone were determined (Table 1). Levels of antibiotics close to what is achievable with standard clinically prescribed treatment dosing were chosen to evaluate persister formation in B. burgdorferi, and we used colony forming unit (CFU) counts to determine viability.
 
 
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21-03-2016
Cohen Foundation przekazala $ +6,5 mln na badania Dr.Lewisa.Dotycza one Boreliozy.
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Lewis i członkowie jego laboratorium odkryli że istnieją podobieństwa między Borrelia  oraz innymi patogenami powodujacymi choroby przewlekłe.Sa to uspione komórki persisters ,na które nie działają antybiotyki. "Konwencjonalne antybiotyki nie moga wiele zrobić, jeśli chodzi o zabijanie persisters", powiedział...
Lewis. "Jedną z rzeczy na ktorych sie skupiamy to dowiedzieć się w jaki sposob pozbyć się persisters".
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lab Lewisa opracowało zabieg o nazwie ,,pulsacyjne podawanie". Kiedy antybiotyk jest podany po raz pierwszy, persisters przetrwa. Naukowcy następnie przerywaja podawanie antybiotyku do czasu wypłukania go przez system, a to powoduje ze persisters się budzi.W tym momencie naukowcy uderzaja ponownie antybiotykami. Oczekuje się, że ta technika bedzie zastosowana na ludziach jeszcze w tym roku.
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lab Lewisa ma również na celu odkrycie nowych leków, które będą bardziej skuteczne przeciwko Borrelia niż te,ktore aktualnie sa dostępne.Lewis jest przekonany, że z pomocą od Fundacji Cohen, lepsze i bardziej skuteczne leczenie choroby z Lyme jest możliwe. "Mamy sukcesy w znalezieniu skutecznych środków leczniczych przy innych chorobach przewlekłych i to doświadczenie pomoże nam rozwiązać problem przewlekłej boreliozy" powiedział Lewis.
 
http://www.northeastern.edu/cos/2016/03/kim-lewis-new-treatments-lyme-disease-grant/
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The Search for Persisters


Lyme disease–causing bacteria can outmaneuver antibiotics in vitro and manipulate the mouse immune system.

 
 
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The Paradox of Chronic Infections – Kim Lewis

What processes take place in a bacteria population under antibiotic treatment? What important discovery made during World War II was forgotten? How can we circumvent the protection mechanisms used by bacteria populations? Professor of Biology at Northeastern University Kim Lewis explains the significance of a rediscovered phenomenon.

North­eastern Uni­ver­sity researchers have found that the bac­terium that causes Lyme dis­ease forms dor­mant per­sister cells, which are known to evade antibi­otics. This sig­nif­i­cant finding, they said, could help explain why it’s so dif­fi­cult to treat the infec­tion in some patients.

Researchers’ discovery may explain difficulty in treating Lyme disease –

This is the first time, we think, that pulse-​​dosing has been pub­lished as a method for erad­i­cating the pop­u­la­tion of a pathogen with antibi­otics that don’t kill dor­mant cells,” Lewis said. “The trick to doing this is to allow the dor­mant cells to wake up.”

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Ciekawe jest to, że doksycyklina jest podawana standardowo przy leczeniu boreliozy.Zaleca tak IDSA i CDC.
Dr.Lewis mówi: "Doksycyklina moze być stosowana jako standardowe leczenie pierwszego rzutu w Lyme, ale nie zabija B. burgdorferi, po prostu ogranicza jej wzrost, pozostawiając resztę pracy dla układu odpornościowego....

http://healthlivetips.org/researchers-finding-new-drugs-in-the-fight-against-lyme-disease/
 
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Nasze wyniki wskazują po terapii B. burgdorferi, 0,001% do 1% komórek może przetrwać śmiertelne dawki różnych antybiotyków in vitro.  Te komórki persister moga przyczynić się do niepowodzenia leczenia u pacjentów z przewlekłą boreliozą. . Przyszłe eksperymenty zmierzają do eliminacji persisters B. burgdorferi. 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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Kim Lewis, "Paradoks przewlekłych zakażeń"
 http://www.northeastern.edu/adc/research/persister-cells/
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Kim Lewis  persister cells:

https://www.youtube.com/watch?v=qXTPiJWhtDg

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Borrelia burgdorferi, the causative agent of Lyme disease, forms drug-tolerant persister cells.
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

13.09-. 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.
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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

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Researchers investigate four promising new treatments for Lyme diseaseMarch 29, 2016 by Thea Singer

http://www.northeastern.edu/news/2016/03/researchers-investigate-four-promising-new-treatments-for-lyme-disease/

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 Superbugs0 8-09- 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.
..

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."
 
https://www.youtube.com/watch?v=plVk4NVIUh8
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Structured Abstract

BACKGROUND

The escalating crisis of multidrug resistance is raising fears of untreatable infections caused by bacterial “superbugs.” However, many patients already suffer from infections that are effectively untreatable due to innate bacterial mechanisms for persistence. This phenomenon is caused by the formation of specialized persister cells that evade antibiotic killing and other stresses by entering a physiologically dormant state, irrespective of whether they possess genes enabling antibiotic resistance. The recalcitrance of persister cells is a major cause of prolonged and recurrent courses of infection that can eventually lead to complete antibiotic treatment failure. Regularly growing bacteria differentiate into persister cells stochastically at a basal rate, but this phenotypic conversion can also be induced by environmental cues indicative of imminent threats for the bacteria. Size and composition of the persister subpopulation in bacterial communities are largely controlled by stress signaling pathways, such as the general stress response or the SOS response, in conjunction with the second messenger (p)ppGpp that is almost always involved in persister formation. Consequently, persister formation is stimulated under conditions that favor the activation of these signaling pathways. Such conditions include bacterial biofilms and hostile host environments, as well as response to damage caused by sublethal concentrations of antibiotics.

http://science.sciencemag.org/content/354/6318/aaf4268

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