Antibiotikų krizė: kaip į ją patekome ir kur išeitis?...fluoroquinolone resistance Some types...
Transcript of Antibiotikų krizė: kaip į ją patekome ir kur išeitis?...fluoroquinolone resistance Some types...
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Vytauto Didžiojo universitetas
Antibiotikų krizė: kaip į ją
patekome ir kur išeitis?
Rimantas Daugelavičius
VDU Biochemijos ir biotechnologijų katedra
2007–2013 m. Žmogiškųjų išteklių plėtros veiksmų programos
3 prioriteto „Tyrėjų gebėjimų stiprinimas“
VP1-3.1-ŠMM-05-K priemonės „MTTP tematinių tinklų,
asociacijų veiklos stiprinimas“
projektas „Lietuvos Biochemikų draugijos potencialo kurti
žinių visuomenę didinimas“
(Nr. VP1-3.1-ŠMM-05-K-01-022)
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7 balandžio - Pasaulio sveikatos diena (PSO (WHO), įkurtos 1948 m., gimtadienis)
Kiekvienais metais švenčiama Pasaulio sveikatos diena būna teminė. Taip akcentuojami PSO veiklos prioritetai ir
akcentuojamos didžiausio rūpesčio sritys
2011
Antimicrobial resistance: no action today no cure tomorrow
2012
Ageing and health: Good health adds life to years
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Penicilinas, pirmasis gamtinis
antibiotikas, atrastas 1928 m.
1881-1955
Alexander Fleming
Už penicilino atradimą ir
įdiegimą į medicininę praktiką,
Ernst Chain, Howard Florey ir
Alexander Fleming 1945 gavo
Nobelio premiją Medicinos
srityje.
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According to the 2008 study, every year at least 25 000 patients
in the European Union alone die from an infection caused by
multidrug-resistant bacteria and estimated additional health-
care costs and productivity losses are at least 1.5 billion Euros.
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Antimicrobial resistance (AMR) is resistance of a
microorganism to an antimicrobial medicine to which it was
previously sensitive.
Methicillin-resistant Staphylococcus aureus (MRSA)
Vancomycin-resistant enterococci (VRE)
Extended-spectrum b-lactamase (ESBL)-producing
Enterobacteriaceae (examples of common Enterobacteriaceae
are Escherichia coli and Klebsiella pneumoniae)
Carbapenemase-producing Enterobacteriaceae (e.g. Klebsiella
pneumonia)
Multidrug-resistant Pseudomonas aeruginosa
Clostridium difficile
Examples of common multidrug-resistant bacteria
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Microbe
Diseases caused
Drugs resisted
Staphylococcus aureus bacteremia (blood infection), pneumonia,
surgical-wound infections
Chloramphenicol, Rifampin, Methicillin,
Ciprofloxacin, Clindamycin, Erythromycin,
Beta-lactams, Tetracycline, Trimethoprim
Streptococcus pneumoniae meningitis, pneumonia, otitis media (ear
infection)
Aminoglycosides, Penicillin,
Chloramphenicol, Erythromycin,
Trimethoprim- Sulfamethoxazole
Mycobacterium tuberculosis tuberculosis Aminoglycosides, Ethambutol, Isoniazid,
Pyrazinamide, Rifampin
Haemophilus influenzae epiglottitis, meningitis, otitis media,
pneumonia, sinusitis
Beta-lactams, Chloramphenicol,
Tetracycline, Trimethoprim
Enterobacteriaceae (e.g. Klebsiella
pneumonia, Escherichia coli, Salmonella)
bacteremia, pneumonia, urinary-tract or
surgical- wound infections, diarrhea
Aminoglycosides, Beta-lactams,
Chloramphenicol, Trimethoprim
Enterococcus bacteremia, urinary-tract or surgical-
wound infections
Aminoglycosides, Beta-lactams,
Erythromycin, Vancomycin
Neisseria gonorrhoeae gonorrhea Beta-lactams, Penicillin, Spectinomycin,
Tetracycline
Pseudomonas aeruginosa bacteremia, pneumonia, urinary-tract
infections
Aminoglycosides, Beta-lactams,
Ciprofloxacin, Tetracycline, Sulfonamides
Bacteroides septicemia, anaerobic infections Penicillin, Clindamycin
Shigella dysenteriae severe diarrhea Ampicillin, Trimethoprim-Sulfamethoxazole,
Tetracycline, Chloramphenicol
Ten common antibiotic-resistant bacteria
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Four main mechanisms resistance to antimicrobials are:
1.Drug inactivation or modification: for example, enzymatic deactivation of
penicillin G in some penicillin-resistant bacteria through the production of β-
lactamases
2.Alteration of target site: for example, alteration of PBP—the binding target
site of penicillins—in MRSA and other penicillin-resistant bacteria
3.Alteration of metabolic pathway: for example, some sulfonamide-resistant
bacteria do not require para-aminobenzoic acid (PABA), an important precursor
for the synthesis of folic acid and nucleic acids in bacteria inhibited by
sulfonamides, instead, like mammalian cells, they turn to using preformed folic
acid.
4.Reduced drug accumulation: by decreasing drug permeability and/or
increasing active efflux (pumping out) of the drugs across the cell surface
'Persister' bacterial cells are temporarily hyper-resistant to all antibiotics at
once. They are able to survive (normally) lethal levels of antibiotics without
being genetically resistant to the drug. These cells are a significant cause of
treatment failure yet the mechanism behind the persistence phenomenon is still
unclear.
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There are three known mechanisms of
fluoroquinolone resistance
Some types of efflux pumps can act to decrease intracellular quinolone
concentration.
In Gram-negative bacteria, plasmid-mediated resistance genes produce
proteins that can bind to DNA gyrase, protecting it from the action of
quinolones.
Finally, mutations at key sites in DNA gyrase or topoisomerase IV can
decrease their binding affinity to quinolones, decreasing the drug's
effectiveness. Research has shown the bacterial protein LexA may play a
key role in the acquisition of bacterial mutations giving resistance to
quinolones and rifampicin.
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Staphylococcus aureus
Found on the mucous membranes and the human skin of around a third of the
population, it is extremely adaptable to antibiotic pressure.
It was one of the earlier bacteria in which penicillin resistance was found—in 1947,
just four years after the drug started being mass-produced. Methicillin was then the
antibiotic of choice, but has since been replaced by oxacillin due to significant kidney
toxicity.
Methicillin-resistant Staphylococcus aureus (MRSA) was first detected in Britain in 1961,
and is now "quite common" in hospitals. MRSA was responsible for 37% of fatal cases
of sepsis in the UK in 1999, up from 4% in 1991. Half of all S. aureus infections in the
US are resistant to penicillin, methicillin, tetracycline and erythromycin.
This left vancomycin as the only effective agent available at the time. However, strains
with intermediate (4-8 μg/ml) levels of resistance, termed glycopeptide-intermediate
Staphylococcus aureus (GISA) or vancomycin-intermediate Staphylococcus aureus
(VISA), began appearing in the late 1990s. The first identified case was in Japan in
1996, and strains have since been found in hospitals in England, France and the US.
The first documented strain with complete (>16 μg/ml) resistance to vancomycin, termed
vancomycin-resistant Staphylococcus aureus (VRSA) appeared in the United States in
2002.
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Meticilinui atsparus St. aureus Europoje 2006
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MRSA paplitimas
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E. coli on the March
Toxic strains of a common gut microbe
are multiplying
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The first rule of
antibiotics is try not to
use them, and the
second rule is try not
to use too many of
them.
—Paul L. Marino, The
ICU Book
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Until recently such completely resistant bacteria have only been found in
hospitals. Now we are starting to see virtually or totally pan-resistant bacteria spilling
into the community.
The outbreak of resistant strains of Escherichia coli – a common cause of food
poisoning – carrying a gene called NDM1 (New Delhi metallo-β-lactamase) in India in
2010, which spread to other countries, is a case in point.
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A shrinking field of research into new antibiotics, which are slow and expensive to
develop. All currently used antibiotics were introduced between 1940 and
1962 then, after a gap of 38 years, the new class of oxazolidinones followed in
2000.
During the past three decades, only two new classes of antibiotics have
been found, and sales shrank to $14.4bn in 2010 from $16.1bn in 2005.
With many existing antibiotics having been around for decades and available as
generics, the bar for prices is low.
Antibiotics have a poor return on investment because they are taken for a short
period of time and cure their target disease. In contrast, drugs that treat chronic
illness, such as high blood pressure, are taken daily for the rest of a patient’s life
If a new antibiotic is approved, it tends to be kept as a last line of defence
against the most serious infections, minimising the sales opportunity. The practice
of reserving new products exclusively as a treatment of last resort for the worst
bacteria significantly reduces the opportunity for companies to recoup their
research and development costs.
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That’s why many companies have stopped developing antibiotics altogether. Only five
major pharmaceutical companies – albeit five of the biggest – GlaxoSmithKline,
Novartis, AstraZeneca, Merck and Pfizer, still had active antibacterial discovery
programmes in 2008
Adding to the grim picture, a comprehensive study of antibiotic development, covering
innovative, small firms, as well as pharma giants, found in 2008 that only 15 antibiotics
of 167 under development had a new mechanism of action with the potential to meet
the challenge of multidrug resistance. Most of those were in the early phases of
development.
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New targets for antibacterials:
Aminoacyl-tRNA-synthase
Polypeptide deformylase
Fatty acid synthesis enzymes
Virulence factors
Outer membrane proteins
The IDSA launched its "Bad Bugs, No Drugs," campaign in 2010 which aims to get
10 new antibiotics on the market by 2020, and has been active in lobbying for
increased government research in antibiotics.
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Šiuo metu tikrinami 9 intraveniniai junginiai, aktyvūs prieš Gramneigiamąsias
bakterijas: 1 β-laktamazės slopiklis šlapimo takų infekcijoms (III fazė), ir 8 junginiai (3 β-
laktamazės slopiklių kombinacijos, II fazė odos bakterinių infekcijų gydymui). Dar trys
junginiai yra I klinikinių arba priešklininkinių tyrimų fazėje.
Tik keletas iš 9 junginių yra aktyvūs prieš blogiausius patogenus, tokius, kaip
Acinetobacter baumannii ir Pseudomonas aeruginosa. Tik du iš II fazėj tikrinamų
junginių turi naują veiklos mechanizmą. Vienas yra pernašos RNR sintazės slopiklis, o
antrasis - peptidomimetikas. Nauji mechanizmai – papildomi saugumo rūpesčiai.
Kibdelomicin - "the first truly novel bacterial type II topoisomerase inhibitor with
potent antibacterial activity discovered from natural product sources in more than six
decades". was dug out from an organism in a sample from the Central African Republic
by a complicated but useful screening protocol
Fidaxomicin is the first of a new class of antibiotics called macrocycles; it’s a narrow-
spectrum drug aimed specifically at Clostridium difficile, the bacterial, toxin-producing,
potentially fatal infection of the gut that occurs when broad-spectrum antibiotics have
killed off the other populations of bacteria that normally live in the intestines.
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0
5
10
15
20
25
30
35
40
1992-1993 1994-1995 1996-1997 1998-1999 2000-2001
%
0% 0%
6%
27%
37%
DVA pasižyminčių S. enterica ser. Typhimurium
paplitimas Centrinėje ir Rytų Europoje
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Išmetimo siurblių tipai
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RND šeimos siurblio AcrAB-TolC struktūra
40
Citoplazma
Vaistas
Periplazma
PM
IM
Citoplazma
Vaistas
Periplazma
PM
IM
Pos, PNAS, 2009
Citoplazma
Vaistas
Periplazma
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Bakterijose esančių siurblių klasifikacija
Šeima Energijos
šaltinis
Substrato
specifiškumas
Aminorūgščių
skaičius
Bakterijų gentys
ABC
didšeimė
ATP Specifinis ir
nespecifinis
varijuoja Escherichia
Lactobacillus
Staphylococcus
SMR
šeima
Protonovaros
jėga
Nespecifinis ~ 110 Bacillus
Escherichia
Staphylococcus
MFS
didšeimė
Protonovaros
jėga
Specifinis ir
nespecifinis
400-600 Lactobacillus
Staphylococcus
Bacillus
Echerichia
Streptococcus
MATE
šeima
Protonovaros
jėga
Nespecifinis > 1000 Haemophilus
Vibrio
Bacillus
RND
šeima
Protonovaros
jėga
Nespecifinis > 1000 Escherichia
Pseudomonas
Neisseria
Haemophilus
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Išmetimo sistemos E. coli
• Chromosomoje koduojami siurbliai: 19 MFS, 3 SMR, 7 RND,
7 ABC, 1 MATE
20 siurblių gali pernešti ląstelei toksiškas/antibiotikų
molekules
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P. aeruginosa siurbliai
Siurblys Substratai
MexAB-OprM β-laktamai, fluorochinolonai, tetraciklinas, makrolidai, chloramfenikolis, pesticidai
MexCD-OprJ β-laktamai, fluorochinolonai, tetraciklinas, makrolidai, chloramfenikolis, pesticidai
MexEF-OprN Fluorochinolonai, chloramfenikolis, pesticidai
MexXY-OprM
Aminoglikozidai, β-laktamai, fluorochinolonai,
tetraciklinas, makrolidai, chloramfenikolis
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S. enterica siurbliai:
Išmetimo sistema Substratai
TetA (MF) Tetraciklinas, TPP+;
AcrAB-TolC (RND) Tetraciklinas, TPP+,
chloramfenilokis, chinolonai ir kt.
MdfA (MF) Tetraciklinas, chloramfenikolis,
makrolidai, aminoglikozidai ir kt.
MdsABC (RND) TPP+, novobiocinas ir kt.
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ABC transporterių mechanizmai
• L. lactis LmrA 3D modelis – flipazė
• S. aureus Sav1866 – “vacuum cleaner”
Ecker G.F et al. Mol Pharmacol. 2004; 66:1169-1179. Schuldiner S. Nature. 2006; 443:156-157.
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Baltymas P-gp dar žinomas kaip
ABCB1, MDR1, PGY1
ATP-binding cassette sub-family B
member 1
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Problema: Atsparumas antibiotikams dėl jų išmetimo iš ląstelės
Galimi sprendimo būdai: – Siurblių nepernešami vaistai
– Pernašos siurblių slopinimas
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Potencialūs DVA siurblių slopikliai:
1. Augalinės kilmės junginiai: barberinas, eteriniai aliejai, izoflavonoidai, žaliosios arbatos ekstraktas, gervuogių sultys, raudonėlio, vynuogių sėklų ekstraktai
2. Kiti vaistai
Žinomos farmakokinetinės savybės, pašaliniai poveikiai
3. Naujai susintetinti junginiai
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DVA slopiklių panaudojimo problemos
• Būtinai kombinuojama dviejų vaistų terapija:
– DVA substrato ir slopiklio farmakokinetinės savybės
turi būti suderintos
• Kiekvienas MDR slopiklis yra dar vienas naujas
vaistas. Neaiški sąveika su mūsų organizmo
ląstelių siurbliais
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DVA slopikliai
• Sukuria „kamštį” išorinės
membranos kanale;
• Sukelia konkurenciją
siurblio išmetamam vaistui;
• Apsunkinti trikomponentės
sistemos susijungimą
• Trikdo siurblio aprūpinimą
energija
• Keičia membranos savybes
Slopiklių veikimo mechanizmai
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Laboratoriniams tyrimams naudojami slopikliai
• Rezerpinas
• Chlorpromazinas
• Fenilalanilarginino-β-naftilamidas (PAβN)
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Populiariausi DVA siurblių slopikliai, naudojami
laboratoriniuose tyrimuose
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Ind
ikato
rin
is e
lektr
od
as
Paly
gin
am
asis
ele
ktr
od
as
Potenciometrinių matavimų sistema ir
indikatoriniai jonai
TPP+
Et+
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Polymyxin B (PMB)
Polymyxins – closely related cyclic peptides
Polymyxin B = Polymyxin B1 + B2
Colistin (polymyxin E) is a mixture of colistin A and B
Source – Bacillus polymyxa (1947)
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Antibacterial peptides
I
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discovery of bacteriophages by Frederick
Twort and Felix d'Hérelle[8] in 1915 and
1917
Frederick Twort Felix d'Hérelle
http://en.wikipedia.org/wiki/Frederick_Tworthttp://en.wikipedia.org/wiki/Frederick_Tworthttp://en.wikipedia.org/wiki/Felix_d'H%C3%A9rellehttp://en.wikipedia.org/wiki/Felix_d'H%C3%A9rellehttp://en.wikipedia.org/wiki/Phage_therapyhttp://en.wikipedia.org/wiki/Frederick_Tworthttp://en.wikipedia.org/wiki/Frederick_Tworthttp://en.wikipedia.org/wiki/Felix_d'H%C3%A9rellehttp://en.wikipedia.org/wiki/Felix_d'H%C3%A9relle
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http://www.sp.uconn.edu/~terry/229sp03/lectures/viruses.html
Bacteriophage infection cycle
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http://www.youtube.com/watch?v=zGSSDJhHgp0&feature=related
http://www.youtube.com/watch?v=rzdwfwuVWUU&feature=relatedhttp://www.youtube.com/watch?v=zGSSDJhHgp0&feature=related
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This figure, based on the data in the 1943 mouse studies of Rene Dubos, provides
significant insight into why phage therapy works well even in treating infections that
antibiotics can't reach. When he injected the mice intraperitoneally with 109 phages, they
quickly appeared in the blood stream, entering the brain, but they were rapidly cleared.
However, if the mice were also injected intracerebrally with Shigella dysenteriae,
the host for these phages, then 46/64 of the mice survived (as compared with 3/84
in the absence of appropriate viable phage) and the brain level of phage climbed to over
109 per gram. Once the bacteria were cleared, phage levels dropped below detection
limits.
Bacteriophage. 2011 Mar-
Apr; 1(2): 66–85.
doi: 10.4161/bact.1.2.15845
http://www.ncbi.nlm.nih.gov/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=An external file that holds a picture, illustration, etc.Object name is bact0102_0066_fig001.jpg [Object name is bact0102_0066_fig001.jpg]&p=PMC3&id=3278644_bact0102_0066_fig001.jpg
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An electron micrograph of bacteriophages
attached to a bacterial cell. These viruses
are the size and shape of coliphage T1
The direct human use of phages is likely to
be very safe; suggestively, in August
2006, the United States Food and Drug
Administration approved spraying meat
with phages. The approval was for
ListShield (a phage preparation targeted
against Listeria monocytogenes) created
by Intralytix. This was the first approval
granted by the FDA and UDSA for a
phage-based food additive.
There are no non-toxic antibiotics to treat
some bacteria such as multiple-resistant
Klebsiella pneumoniae, but killing of the
bacteria via intraperitoneal, intravenous
or intranasal of phages in vivo has been
shown to work in laboratory tests.
Enzobiotics are a new development at
Rockefeller University that create enzymes
from phage.
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The first controlled clinical trial of a therapeutic bacteriophage preparation
showed efficacy and safety in chronic otitis because of chemo-resistant P.
aeruginosa.
Phage therapy is flawed for a number of reasons:
1. Bacteria can and frequently do become resistant to phage.
2. Phage are usually species and sometimes strain specific. In a clinicial setting,
infections can be of mixed species and finding out the infecting species, let alone the
strain, takes time.
3.Phage can generate an antibody response, which has the potential render them
useless.
4.Phage can shuttle genes encoding antibiotic resistance and virulence factors
from one bacteria to another.
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Fagu Bam35 infekuotų B. thuringiensis ląstelių lizės
eigos priklausomybė nuo suspensijops aeracijos
intensyvumo
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Ačiū už dėmesį!