bacterial antibiotic resistance development Costs of antibiotic resistance

bacterial antibiotic resistance development

Costs of antibiotic resistance EU estimate: 25, 000 deaths and € 1. 5 billion per year Hospital infections: between € 6000 and € 50, 000 extra per case when compared to susceptible of the same pathogen.

If TIME Magazine says it is important 3

Antibiotic: Compound, made by another cell or organism, that inhibits the growth of microorganisms or kills them. Antimicrobial: Kills microbes, e. g. Triclosan Desinfectant: Kills everything, mixtures of e. g. peracetic acid, hydrogen peroxide, sodium hypochlorite, didecyldimethylammoniumchloride, glutaraldehyde, formaldehyde, etc

Antibiotic / antimicrobial Made by another (micro)organism Very specific Single mechanism 5

History antibiotics 1928 Fleming 1938 Chain and Florey make penicillin useful in humans 1942 First life saving treatment 1944 D-day; enough penicillin for all soldiers 1946 -1960 Wonder drug era; discovery of tetracycline, chloramphenicol, many others

Antibiotics; 5 classes of mechanisms - Interference with cell wall synthesis β-Lactams; glycopeptides - Protein synthesis inhibition (50 Sunits) macrolides, chloramphenicol, clindamycin, quinupristin-dalfopristin, linezolid, (30 Sunits) aminoglycosides, tetracyclines (isoleucyl-t. RNA synthase) mupirocin - Interference with nucleic acid synthesis (DNA) fluoroquinolones; (RNA) rafimpin - Inhibition metabolic pathway: sulfanomides, folic acid analogues - Disruption membrane structure: polymixins, daptomycin

First antibiotic in humans: penicillin 1942 First case of resistance 1947

Early sixties: First signs of resistance Late sixties – early seventies: analogs Eighties and nineties: Resistance crisis reduction of agricultural use, restrictive use in humans 20? ? Postantibiotic ? ?

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Mechanisms for antibiotic resistance 1: Enzymatic inactivation 2: Go around the affected pathway 3: Change the target 4: Pump the drug out 5: Break down the antibiotic outside of the cell. Example: (extended spectrum) beta lactamases

Ways to become resistant: Destroy the antibiotic The beta-lactamase enzyme opens the beta-lactam ring and thereby inactivates the antibiotic 12

Go around the problem Alternative pathway A* S A B* B C* X C D* D P The step B to C is is blocked by an antibiotic 13

Change the target antibiotic 14 target antibiotic new target

The cell pumps the antibiotic out The environment Inside of the cell = 15

Entrance of the antibiotic is blocked The environment Antibiotic molecules 16 ≠ Inside of the cell

Antibiotics are natural compounds, therefore resistance genes have been present in nature for millions of years. Antibiotics are produced by microorganisms; The producer must be resistant against the compound it produces

There are three ways around the barrier 18

There are three ways to acquire resistance: 1) Physiological adaptation by converting existing cellular elements such as pumps 2) Mutating genes that code for proteins that in the new version cause resistance 3) Horizontal gene transfer e. g. of plasmids carrying resistance genes

Examples of aquisition of resistance: - Altering house keeping genes (Adaptation) Penicillin resistance in E. coli - Changing the target (Mutation) fluoroquinolone resistance in Pseudomonads - Transfer of resistance genes already present in nature Tetracycline resistance, ESBL’s Selection multiplies the effects

Once an organism is resistant against one antibiotic, the chance that it will become resistant against more increases Acquisition of resistance also selects for the ability to pick up resistance, either by changing house keeping genes, or by gene transfer. Multidrug resistance becomes very common

Typically focus is on a single gene 22

Alekshun and Levy, 2007

Goal of SILS research To create a laboratory model for the acquisition, transfer and loss of resistance to various antimicrobial agents (e. g. amoxicillin, tetracycline and enrofloxacin) in several drug-bug combinations chosen for different mechanisms Basic question: how much usage and which manners of dosage lead to how much resistance?

Molecular level: Follow genetic changes, either in plasmid or in genomic DNA Gene transfer versus mutations Calculate chance of event happening under different selection pressures

Conclusions: Central cellular mechanism is crucial for development of resistance against bactericidals Essential role for genes involved in regulation of transcription Response depends on class of antibiotic in combination with environment 26

Can the observed increase in resistance in microbiota of livestock during exposure to antimicrobials be explained by de novo development of resistance, or must exchange of genetic information be involved? 27

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Transfer of plasmids Driving factors 30

Transfer experimental setup 64 mg/l tetracycline 320 ml E. coli MG 1655 +YFP E. coli 23: 06 ~2. 25 g/l DW + indole + chloramp + YFP +Ind +Cl + integron + tet. Res. + PCR 5’-3’ CS Mix +Ind +Cl

Conclusions plasmid transfer experiments • Plasmid transfer occurs in both presence and absence of selective pressure • A minimal density is needed for effective transfer • Plasmid transfer is increased when nonlethal levels of antibiotics are used • Plasmid transfer is dependent on growth rates • Energy is needed for plasmid transfer 32

ESBL’s Extended spectrum beta-lactamases exoenzymes that break down penicillins and cephalosporins Also confer resistance to other antibiotics, such as tetracyclin and fluoroquinolones Located on plasmids. CTX-M is associated with the agricultural sector

Almost all ESBL producers are multi-resistant

Superbugs, hospital bugs MRSA Meticillin Resistant Staphyloccus aureus VRE Vancomycin resistant Enterococcus and Multi-drug resistant (MDR) Acetinobacter sp. Partly selected for by tetracycline and avoparcin in the agricultural sector 2003 “Danish experiment” concludes that ban on growth promoters has lots of advantages, few disadvantages

Avoparcin Vancomycin Triclosan Structural relationship causes cross-res

Resistance in 480 soil derived bacteria

Resistance in soil bacteria

Agricultural use of antibiotics Flock administration, mixed in feed Use as “growth promoter” Growth promoters only effective in “dirty” farms Estimated costs of ban on growth promoters: US $ 5 -10 (1999) person per year (US) Use as growth promoter banned in the EU in 2006

What did we learn that is useful for the Food Safety Authority and public health in general? 1) The greatest risk for both de novo development and for transfer of resistance occurs at exposure to low concentrations of antibiotics. Short treatment at high doses is better than longer therapy at low doses. 2) The limited ecological range of resistant strains can be used to design “smart” measures, such as treating a stable with salt or mildly acid water, to boost susceptible strains over their resistant counterparts. 40

41 Picture taken in a Subway restaurant in NYC