Microbial resistance to antimicrobial agents

Prof. Beata M. Sobieszczańska

Department of Microbiology

University of Medicine

What is a drug resistance?

Antimicrobial resistance is the ability of a microorganism to survive and multiply in the

presence of an antimicrobial agent that would normally inhibit or kill this species of

microorganism

Bacterial resistance to antimicrobials

Intrinsic resistance

Bacteria may be resistant because either:

• lack of affinity of the drug for the bacterial target

• inaccessibility of the drug into the bacterial cell

•extrusion of the drug by chromosomally encoded active exporters

• innate production of enzymes that inactivate the drug

Enterococci

Enterococci are inherently resistant to:

cephalosporins

lincozamides

polymyxin B

low concentrations of aminoglycosides

TMP-SMX

ORGANISMS NATURAL RESISTANCE AGAINST: MECHANISM

Anaerobic bacteria AminoglycosidesLack of oxidative metabolism to drive uptake of aminoglycosides

Aerobic bacteria MetronidazoleInability to anaerobically reduce drug to its active form

GP Aztreonam (β-lactam)Lack of penicillin binding proteins (PBPs) that bind and are inhibited by this β-lactam antibiotic

GN VancomycinLack of uptake resulting from inability of vancomycin to penetrate outer membrane

Klebsiella spp. Ampicillin (β-lactam)Production of enzymes (β-lactamases) that destroy ampicillin before the drug can reach the PBP targets

Stenotrophomonas Imipenem (β-lactam)Production of enzymes (β-lactamases) that destroy imipenem before the drug can reach the PBP targets.

P. aeruginosaSulfonamides, trimethoprim, tetracycline, or chloramphenicol

Lack of uptake resulting from inability of antibiotics to achieve effective intracellular concentrations

Enterococci

AminoglycosidesLack of sufficient oxidative metabolism to drive uptake of aminoglycosides

All cephalosporinsLack of PBPs that effectively bind and are inhibited by these β-lactam antibiotics

Acquired resistance

Mutations

Horizontal gene transfer = genes pass from a resistant strain to a non-resistant strain conferring resistance on the latter

The introduction of an antibiotic into the bacterial environment acts as a selective pressure

Acquired resistance - mutation

Horizontal gene transfer

Conjugation

• Transmission of resistance genes via plasmid exchange

• Plasmids that they can pass to other bacteria during conjugation

• (R plasmids carry r-genes)

• This type of acquisition allows resistance to spread among a population of bacterial cells much faster than simple mutation

Transduction

A virus serves as the agent of transfer

between bacterial strains

Transformation

DNA released from a bacterium is picked

up by a new cell

Biochemical mechanisms of bacterial resistance:

1. Production of enzymes which detoxify or modulate the activity of the antibiotic

2. Alteration of the target site to reduce or block binding of the antibiotic

3. Prevention transport of the antimicrobial agent

4. Development of an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent

β-lactamases = bacterial enzymes

•β-lactamase enzymes can destroy the β-lactam ring of penicillins through hydrolysis

•without a β-lactam ring penicillins are ineffective against the bacteria

• Produced by many GP and GN bacteria

bacterial β-lactamases

Penicillinases

Cephalosporinases

ESBL

MBL

AmpC

Extended-Spectrum β-Lactamases (ESBL)

β-lactamases conferring resistance to the penicillins, cephalosporins & monobactams but

not to carbapenems & cephamycins which are inhibited by β-lactamase inhibitors (BLIs)

Produced by GN rods

Cephamycins (2nd gen. of cephalosporins): cefoxitin, cefotetan, cefmetazole

Extended-spectrum β-lactamases (ESBL)

Genes encoding for ESBL are frequently located on plasmids (spread via HGT) which also carry resistance genes for aminoglycosides, tetracycline, TMP-SMX, fluoroquinolones

Clinical implications:

treatment failure

increased morbidity & mortality

outbreaks

Metallo-β-Lactamases (Carbapenemases ) MBL & KPC

• Hydrolyse virtually all β-lactams

• They are resistant to BLIs but sensitive to aztreonam

• Mediate broad spectrum β-lactam resistance

• Present on plasmids

• Genes are continuously spreading

• Produced by GN rods: Pseudomonas, Klebsiella pneumoniae, E. coli, Proteus mirabilis, Enterobacter

Metallo-β-Lactamases KPC

• KPC – Klebsiella pneumoniae carbapenemase – isolates resistant to many antimicrobials also aminoglycosides and quinolones

• Genes encoding KPC are on mobile genetic elements e.g. plasmids

• KPC strains – multi-resistant

• First combination of carbapenem with BLI –meropenem+vaborbactam active against KPC-positive Klebsiella

AmpC-type β-Lactamases

• Encoded chromosomally (but may also be on plasmids) among GN rods:

• Serratia, Pseudomonas/Proteus, Acinetobacter, Citrobacter, Enterobacter = SPACE

• Hydrolyse broad spectrum cephalosporins + cephamycins

• They are not inhibited by BLIs

• Often inducible by e.g. cephalosporins

• Hallmark phenotypic pattern: these rods appear to be susceptible to 3rd gen. cephalosporins but resistance can develop upon β-lactam exposure

Aminoglycoside modifying enzymes:

N-Acetyltransferases (AAC) – catalyses acetyl CoA-dependent acetylation of an amino group

O-Adenyltransferases (ANT) – catalyses ATP-dependent adenylation of hydroxyl group

O-Phosphotransferases (APH) – catalyses ATP-dependent phosphorylation of a hydroxyl group

GP and GN bacteria resistance to aminoglycosides (phosphorylation, adenylation, acetylation)

Enterobacteria Resistance to chloramphenicol - acetylation

EnterococciSynergistic combination therapy

cell wall active agent (β-lactam/glycopeptides) + high concentration of aminoglycoside

often provides effective therapy (treatment of endocarditis caused by enterococci)

HLAR (High Level of Aminoglycoside Resistance) – enterococci that acquired genes encoding aminoglycoside inactivating enzymes

The synergism of aminoglycosides with cell wall active agent is lost

Biochemical mechanisms of bacterial resistance:

1. Production of enzymes which detoxify or modulate the activity of the antibiotic

2. Alteration of the target site to reduce or block binding of the antibiotic

3. Prevention transport of the antimicrobial agent

4. Development of an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent

Resistance to β-lactams

PBP (Penicillin Binding Proteins = transpeptidase enzyme) alterations:

Streptococcus pneumoniae resistant to penicillin PRP(Penicillin Resistant Pneumococci)

MRSA (Methicillin Resistant Staphylococcus Aureus)

Listeria monocytogenes, gonococci - resistant to β-lactams

Resistance to glycopeptides & ampicillin

GRE (Glycopeptide Resistant Enterococci)

VRE (vancomycin resistant enterococci)

Ampicillin resistant enterococci (altered PBP)

M. tuberculosis – changes in RNA polymerase

Resistance to rifampin

Resistance to macrolides

Resistance to Macrolide, Lincosamide & Streptogramin B antibiotics (MLSB phenotype) –staphylococci, streptococci

Many GN bacteria - alterations in subunits of DNA gyrase

GP bacteria (pneumococci, staphylococci) - alteration in topoisomerase IV

Resistance to fluoroquinolones

Biochemical mechanisms of bacterial resistance:

1. Production of enzymes which detoxify or modulate the activity of the antibiotic

2. Alteration of the target site to reduce or block binding of the antibiotic

3. Prevention transport of the antimicrobial agent

4. Development of an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent

Strategy 1: Prevention of the antimicrobial from reaching its target by reducing its ability to penetrate into the cell = prevention access

Change in the number or characters (size, selectivity) of porin channels in the outer membrane of a Gram-negative cell wall

Strategy 2: Eliminating antimicrobial agents from the cell by expulsion using efflux pumps

Some bacteria possess membrane proteins that act as an export or efflux pump for antimicrobials – some of them selectively extrude specific antibiotics (macrolides, lincozamides, streptogramins, tetracyclines) whereas others expel a variety of structurally diverse antimicrobials

Examples – prevention access:

1. Pseudomonas aeruginosa – imipenem

2. Enterobacter aerogenes – imipenem

3. Vancomycin resistant S. aureus - thickened cell wall trapping vancomycin

GISA (Glycopeptide Intermediate Resistant Staphylococcus Aureus)

VISA (Vancomycin Intermediate Resistant Staphylococcus Aureus)

VRSA (Vancomycin Resistant Staphylococcus Aureus)

4. Many GN bacteria – aminoglycosides, quinolones

Examples – efflux pumps:

1. enterobacteria – tetracyclines, chloramphenicol

2. staphylococci – macrolides, streptogramins

3. staphylococci, pneumococci - quinolones

Biochemical mechanisms of bacterial resistance:

1. Production of enzymes which detoxify or modulate the activity of the antibiotic

2. Alteration the target site to reduce or block binding of the antibiotic

3. Prevention of transport of the antimicrobial agent

4. Development of an alternate metabolic pathway to by-pass the metabolic step being blocked by the antimicrobial agent

Overcoming drugs that

resemble substrates and

tie-up bacterial enzymes

Production of greater amounts of the limited enzyme that is being tied up or inactivated by the

antibiotic

Mode of action

Prevent cross-linking

Resistance

• Enzymatic destruction of β-lactam ring

• Target (PBP) modification

• Reduced intracellular accumulation

• Target modification - productionof false targets

Target & bind PBP

Glycopeptides

β-lactams

Quinolones

Target DNA gyrase and topoisomerase IV = inhibit DNA supercoiling

• Target modification

• Reduced intracellular accumulation

Mode of action Resistance

• Enzymatic modification

• Target modification

• Reduced uptake

• Target modification

• Reduced intracellular uptake

Target & bind 30S ribosomal subunit = protein inhibition

Macrolides

Aminoglycosides

Tetracyclines • Target modification

• Reduced intracellular accumulation

Target & bind 50S ribosomal subunit = protein inhibition

Target & bind 30S ribosomal subunit = protein inhibition

Mode of action Resistance

• Target modification

• Target modification

Target RNA polymerase = block RNA synthesis

Sulphonamides

Rifamycins

Target dihydropteroate synthase = inhibition folic acid synthesis

Examples

Altered target (PBP)

Mechanism of resistance

Resistance of staphylococci to penicillin

Resistance of enterobacteria to penicillins, cephalosporins, monobactams ESBL

Resistance of staphylococci to methicillin and oxacillin MRSA

Enzymatic destruction

(β-lactamases)

β-lactams

Resistance of Enterobacter, Klebsiella and Pseudomonas to imipenem

Decreased uptake

(porin channel formation is decreased)

Examples

Altered target

alteration in the molecular structure of cell wall precursor

Mechanism of resistance

Resistance of enterococci to vancomycin, teicoplanin

VRE, GRE

Glycopeptides

Examples

Altered targetmodification of ribosomal proteins or of 16S rRNA

Mechanism of resistance

Resistance of many GN and GP bacteria to aminoglycosides

e.g. enterobacteria, enterococci

Resistance of many GN bacteria to aminoglycosides

Enzymatic modification

Aminoglycosides

Resistance of Mycobacterium to streptomycin

Decreased uptake

(change in number and character of porin channels)

Examples

Altered targetchanges in DNA gyrase subunits

Mechanism of resistance

Resistance of GN bacteria and staphylococci (efflux only) to various quinolones

Fluoroquinolones

Resistance of GN and GP to various quinolones

Decreased uptake

(alterations in the outer membrane – diminished uptake or efflux pump)

• Bacteria are able to resist antimicrobials by: preventing intracellular access, immediately removing antimicrobial substances through efflux pumps, modifying the antimicrobial through enzymatic breakdown, or modifying the antimicrobial targets within the bacterial cell. Successful development of resistance often results from a combination of two or more of these strategies

• Antimicrobial resistance traits are genetically coded and can either be intrinsic or acquired

• Intrinsic resistance is due to innately coded genes which create natural resistance to a particular antibiotic. Innate resistance is normally expressed by virtually all strains of a particular bacterial species

• Acquired resistance is gained by previously susceptible bacteria either through mutation or horizontally obtained (HGT) from other bacteria possessing such resistance via transformation, transduction, or conjugation. Acquired resistance is limited to subpopulations of a particular bacterial species and may result from selective pressure exerted by antibiotic usage