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)

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

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.

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

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.

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.

Meticilinui atsparus St. aureus Europoje 2006

MRSA paplitimas

E. coli on the March

Toxic strains of a common gut microbe

are multiplying

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

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.

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.

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.

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

Išmetimo siurblių tipai

RND šeimos siurblio AcrAB-TolC struktūra

40

Citoplazma

Vaistas

Periplazma

PM

IM

Citoplazma

Vaistas

Periplazma

PM

IM

Pos, PNAS, 2009

Citoplazma

Vaistas

Periplazma

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

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

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

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.

Baltymas P-gp dar žinomas kaip

ABCB1, MDR1, PGY1

ATP-binding cassette sub-family B

member 1

Problema: Atsparumas antibiotikams dėl jų išmetimo iš ląstelės

Galimi sprendimo būdai: – Siurblių nepernešami vaistai

– Pernašos siurblių slopinimas

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

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

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

Laboratoriniams tyrimams naudojami slopikliai

• Rezerpinas

• Chlorpromazinas

• Fenilalanilarginino-β-naftilamidas (PAβN)

Populiariausi DVA siurblių slopikliai, naudojami

laboratoriniuose tyrimuose

54

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

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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61 http://www.youtube.com/watch?v=rzdwfwuVWUU&feature=related

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

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

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.

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

Ačiū už dėmesį!