Relative or complete lack of effect of antimicrobial against a previously susceptible

microbe

Increase in MIC

Figure 20.20

Horizontal Gene Transfer

A = Transformation; B = Conjugation; C = Transduction

• Enzymatic destruction of drug

• Prevention of penetration of drug

• Alteration of drug's target site

• Rapid ejection of the drug

Clinical resistance vs actual resistance

Resistance can arise by mutation or by gene transfer (e.g.

acquisition of a plasmid)

Resistance provides a selective advantage

Resistance can result from single or multiple steps

Cross resistance vs multiple resistance

› Cross resistance -- Single mechanism-- closely related

antibiotics

› Multiple resistance -- Multiple mechanisms -- unrelated

antibiotics

Resistant organism

MICs of organism are higher than achieved drug

concentrations in tissues

Intermediately resistant

the antibiotic may still be effective but higher

doses should be used

Highly resistant

the antibiotic tissue concentrations are likely not

to exceed MICs of the microorganisms

Terminologies

Intrinsic or natural resistance

G-neg bacteria are resistant to vancomycin (large

molecule)

Tetracyclines are hydrophobic, G-neg bacilli are

resistant

Acquired resistance

Mutations (PBP)

Disseminated by plasmids and transposons

Spontaneous mutations

Types of resistance

Mechanisms of antibiotic resistance

1. Production of enzymes

destroying and modifying AB

ß-lactamases AG modifying

enzymes

2. Decrease of cell

membrane permeability

3. Active efflux of AB from

cell

4. Modification of AB target

sites

Genetics and spread of drug resistance

Viridans Streptococci

S.pneumoniae

S.Epidermidis

S.aureus

E.faecium

S.aureus

Transposon . genes moving from one point to another (jumping genes)

Bacteriophagevirus, infecting bacteria (virus of bacteria)

Integronslice(s) of DNA, cassette of gene that may be entered into

other cell

Plasmidcircular double stranded DNA molecule, located separately

of the chromosomal RNA

Production of enzymes inactivating (destroying)

antibiotics

ß-lactamases

Main mechanism of resistance in ß-lactam

antibiotics

Penicillin-resistant S.aureus

Ampicillin-resistant E.coli

Production of enzymes modifying antibiotics

Aminoglycosides, chloramphenicol

(1) Mechanisms of resistance

Resistance mechanisms: inactivating enzymes (2)

Degrading enzymes will bind to the

antibiotic and essentially degrade it

or make the antibiotic inactive

Blocking enzymes attach side chains

to the antibiotic that inhibit its function.

E.g. ß-lactamases

PBP & ß-lactamase

Serine proteases (PBP) a metalloenzymes (Zn-binding thiole group as

coenzyme)

200 different enzymes e.g. penicillinases, cephalosporinases, ESBL,

AmpC

ESBL - extended spectrum ß-lactamases (broad spectrum of activity);

encoded in plasmids, can be transferred from organism to organism

Production of ß-lactamases: mechanism of

action

Examples

TEM-1 is a

widespread ß-

lactamase of

Enterobacteriaciae

that attacks

Penicillin G and

narrow spectrum

cephalosporins

>50% AmpR

E.coli isolates are

caused by TEM-1

Altered permeability

› Altered influx

Gram negative bacteria

Antibiotics are removed via active efflux pump

Universal efflux pump

specific efflux pump

quinolones, tetracyclines, chloramphenicol

Efflux Mechanisms of resistance

Resistance mechanisms: efflux pump

The efflux pump is a membrane bound protein that

"pumps" the antibiotic out of the bacterial cell

Microbe Library

American Society for Microbiology

www.microbelibrary.org

Altered permeability

› Altered efflux

tetracycline

Microbe Library

American Society for

Microbiology

www.microbelibrary.org

Inactivation

› ß-lactamase

› Chloramphenicol acetyl transferase

Microbe Library

American Society for

Microbiology

www.microbelibrary.org

Modification of target sites

altered PBP (PRSP)

new PBP (MRSE, MRSA)

Modification in ribosomes (macrolideresistant

S.pneumoniae)

Mechanisms of resistance

Altered target site

› Penicillin binding

proteins (penicillins)

› RNA polymerase

(rifampin)

› 30S ribosome

(streptomycin)

Microbe Library

American Society for

Microbiology

www.microbelibrary.org

Modification of AB target sites:

disruption in protein synthesis

VRE . vancomycin-resistant enterococci

70% of E. faecium strains in USA

GISA . glycopeptide intermediately susceptible S.aureus

VISA . vancomycin intermediately susceptible S.aureus

VRSA & VRSE . vancomycin-resistant S.aureus and S.epidermidis

(MIC> 32 mcg/ml; 1st clinical case described in 2002 in USA)

ESBL producing K.pneumoniae . Extended spectrum ß-lactamase

producing K. pneumoniae

PRSP penicillin-resistant S. pneumoniae

Important terms among drug

resistant microorganisms

ß-lactam antibiotics:

penicillins

cephalosporines

carbapenems

Alexander Fleming

P. chrysogenum

(original strain of Fleming)

destroy Staphylococcus aureus

1928

ß-lactam structure is presented in red and blue

Side chain is presented in black

Penicillins

Carbapenems

Cephalosporins

Mechanism of action of ß-lactam antibiotics

1ß-lactam ab

binds to PBP

2. Inhibition of

peptidoglycan

synthesis

3. Cell death

Structure of peptidoglycan

ß-lactams inhibit synthesis of crosslinks

Penicillins

Cephalosporins

Initially isolated form

the mould Cephalosporium

Compared with penicillins:

More resistant to ß-

lactamase hydrolysis

Wider antibacterial spectrum

Improved PK-properties

Resistance to ß-lactam

antibiotics

Resistance to ß-lactam antibiotics

Production of ß-lactamases

Penicillin-resistant S.aureus (>95%) - Synthetic

Penicillins

ESBL K.pneumoniae - IV generation cephalosporins,

carbapenems

Ampicillin-resistant E.coli – cephalosporins

Changes in the structure of PBP

(altered PBP) Penicillin-resistant S.pneumoniae -

larger doses of penicillin

New PBP - MRSA, MRSE . vancomycin

Disruption of bacterial cell wall

Glycopeptides

vancomycin

teicoplanin

Vancomycin: mechanism of action

Mechanism - vancomycin inhibits cross linkage between

peptidoglycan layers

Vancomycin can bind only to D-Ala-D-Ala and not to D-Ala-D-lac

Originally obtained form

Streptomyces orientalis

Active only against G+

bacteria (large molecule

unable to penetrate outer

membrane of G+ bacteria)

Used for treatment of

oxacillin resistant G+

infections

Intrinsic resistance (pentapetide end with D-Ala-D-Lac)

Leuconostoc, Lactobacillus, Pediococcus

Or with D-Ala-D-Ser

Enetrococcus gallinarum, Enetercoccus caselliflavus

Acquired resistance

A thickening of the PG layer, and

Modification of the PG termini from D-Ala--D-Ala to D-Ala--D-lactate

Gene (vanA, B, C, D, G, E) is carried on plasmids & may be

transferred from organism to organism

Importance

VRE - vancomycin resistant E. faecium, E.faecalis

VISA - vancomycin intermediately resistant S.aureus

GISA - glycopeptide intermediately resistant S.aureus

VRSA - vancomycin resistant S.aureus (MIC> 32 µg/ml; 1st

clinical case reported 2002 in US)

Mechanism of Resistance to Vancomycin

Bacitracin (cyclic peptides) is isolated form Bacillus licheniformis Topically applied agent against G+ bacteria

Interferes with the dephoshorylation and recycling of the lipid carrier responsible for moving peptidoglycanprecursors

Polymyxin (cyclic polypeptides) derived from Bacillus polymyxa Interact with the lipopolysaccharides and phospholipids in

the outer membrane and thus increase cell permeability

Mostly active against G- bacilli (G+ bacilli do not have outer membrane)

Activity of antibiotics to bacterial

cell wall

G-positive

G-negative

polypeptides ß-lactamsglycopeptides

Inhibition of protein synthesis

Aminoglycosides

Tetracyclines

Oxazolidones

Chloramphenicol

Macrolides

Clindamycin

Streptogramins

Protein synthesis

Substance binding to 30S subunit

Antibiotics that act at the level of protein

synthesis initiation

Antibiotics that act at the level of the

elongation phase of protein synthesis

Consists of aminosugars that are linked through glycosidic rings

Origin

Streptomyces - streptomycin,

neomycin, kanamycin, tobramycin

Micromonospora - gentamicin, Sisomicin

Synthetic derivates

Amikacin = kanamycin

Netilmycin = sisomycin

Mainly active against G-negative bacteria

Gentamycin

Aminoglycoside: mode of action

AG pass through cell wall,

cytoplasmic membrane to

cytoplasma (mainly of Gbacteria,

no penetration through cytoplasmic

membrane of strepto- and

entrococci)

Bind irreversible to the 30S

subunit of bacterial ribosomes and

block the attachment of the 50S

subunit to the initiation complex

As a result production of

aberrant proteins and misreading

of RNA occurs

Aminoglycoside: mode of action

1. Passage through cytoplasmic membrane of G- bacteria (no penetration

through cytoplasmic membrane of strepto- and enterococci)

2. Binding to 30S subunit

3. Misreading the codon along mRNA

4. Inhibition of protein synthesis

Enzymatic modification (common) of the drug

High level resistance

>50 enzymes identified

Genes encoding resistance located in plasmids

Gene transfer occurs across species

Reduced uptake or decreased permeability of bacterial

cell wall

Resistance in anaerobes (transport through

cytoplasmic membrane depends on anaerobic respiration)

Altered ribosome binding sites (rare)

Microbes bind to multiple sitesLow level resistance

Aminoglycoside resistance

TetracyclinesOrigin

Tetracyclin, oxytetracyclin isolated from Streptomyces

Minocyclin, doxycyclin are synthetic

Broad spectrum bacteriostatic antibiotics

Antibacterial spectrum similar to macrolides (incl. Clamydia,

Mycoplasma, Rickettsia)

Resistance (widespread)

Energy dependent efflux pump (most common)

Alteration of ribosomal target (ribosome protection)

Enzymatic change

The tetracyclines block

bacterial translation by binding

reversibly to the 30S subunit and

distorting it in such a way that the

anticodons of the charged tRNAs

cannot align properly with the

codons of the mRNA

Tetracyclines

Newest class of antibiotics; completely synthetic

Narrow spectrum of activity (G+ bacteria, includingVRE,

MRSA)

G-neg bacteria resistant due to efflux pump

Mode of action: unique mechanism among antibiotics;

interferes with the initiation complex at the 50S ribosome

subunit (V domain of 23S rRNA)

Resistance confers to mutation at 23S rRNA

Resistance is rare; cross-resistance unlikely because 23S

rRNA is encoded by several genes

Oxazolidones: linezolid

Oxazolidones: mode of action

Inhibit the formation of an initiation complex by binding to the 50S

ribosomal subunit (domain V of the 23S rRNA), disrupting the preliminary

phases of protein synthesis

Binds irreversible to peptidyl transferase component of 50S

ribosome and blocks peptide elongation, thus interferes with

protein synthesis

Bacteriostatic antibiotic with broad spectrum of antibacterial

activity

Interferes with the protein synthesis of bone marrow cells

causing aplastic anaemia

Limited clinical use in Western world due to side Effect

Resistance is associated with producing

acetyltransferase which catalyses acetylation of 3-hydroxy

group of chloramphenicol

Chloramphenicol

Macrolides (1)

Erythromycin was derived from Streptomyces erythreus

The basic structure is a lactone ring

14-membered lactone ring . erthromycin, clarithromycin, roxithromycin,

telithromyin (ketolide)

15-membered lactone ring . Azithromycin

16-membered lactone ring . spiramycin, josamycin

Acitivity .

broad spectrum G+ bacteria and some G- bacteria including

Chlamydia, Mycoplasma, Legionella, Rickettsia, Neisseria

Azithromycin, Clarithromycin active against some mycobacteria

Macrolides: mode of action

Blocking Translation during Bacterial Protein

Synthesis

The macrolides bind reversibly to the 50S subunit.

They can inhibit elongation of the protein by the peptidyltransferase, the

enzyme that forms peptide bonds between the amino acids.

erythromycin

Mode of Action of Macrolides in Blocking

Translation during Bacterial Protein

Synthesis

The macrolides bind reversibly to

the 50S subunit.

They can inhibit elongation of the

protein by blocking the translocation

of the ribosome to the next codon on

mRNA

Macrolide resistance

Resistance

Intrinsic resistance- hydrophobic macrolides

have low permeability through outer membrane

(G- bacilli)

Acquired resistance

Ribosomal modification

Efflux pump

Enzyme inactivation

Clindamycin, lincomycin

Family of lincosamide antibiotics originally isolated from

Streptomyces lincolnensis

Mode of action: bind 50S ribosome subunit and blocks

protein elongation

Resistance is related to 23S ribosomal RNA Methylation

Active against staphylococci and G-ve anaerobic bacilli.

No activity against aerobic

Replacement of a sensitive pathway

› Acquisition of a resistant enzyme

(sulfonamides,

trimethoprim)

Molecular Drug Susceptibility Testing

• Genotypic methods: the drug target and nature of

the gene mutation are known

• Usually molecular amplification of target

DNA or RNA followed by some means of detecting mutation in the product.

Molecular methods of drug susceptibility testing 1. Sequencing

Universal and reliable methodExpensive, time-consuming and not suitable for everyday routine testingApplied as reference method to verify results of other tests.

2. PCR-based methods

PCR-Single Strand

Conformation Polymorphism (PCR-SSCP)

Mutations cause alterations in

conformation of single-strand DNA fragments and it is registered in non-denaturizing PAGE

Other molecular methods of drug susceptibility testing:

Molecular beacons

Real-Time fluorescent PCR combines amplification and detection: minimises amplicon contamination

PCR+hybridization

Based on amplification of fragments of genesresponsible for drug resistance development

follwed by hybridization with oligonucleotideprobes immobilized on membranes;

Both commercial kits and in-house macro-arrays have been reported to demonstratehigh sensitivity and specificity

Molecular tests for the detection of resistance to RIF and INH

GenoType® MTBDRplus test procedure

Reaction zones of GenoType® MTBDRplus (examples)

Exposure to sub-optimal levels of antimicrobial

Exposure to microbes carrying resistance

genes

Prescription not taken correctly

Antibiotics for viral infections

Antibiotics sold without medical supervision

Spread of resistant microbes in hospitals due to lack of hygiene

Lack of quality control in manufacture or outdated antimicrobial

Inadequate surveillance or defective susceptibility assays

Poverty or war

Use of antibiotics in foods

Antibiotics are used in animal feeds and sprayed on plants to prevent infection and

promote growth

Multi drug-resistant Salmonella typhi has been found in 4 states in 18 people who ate beef fed antibiotics

Infections resistant to available antibiotics

Increased cost of treatment

Methicillin-Resistant Staphylococcus aureus

Most frequent nosocomial (hospital-acquired) pathogen

Usually resistant to several other antibiotics

Speed development of new antibiotics

Track resistance data nationwide

Restrict antimicrobial use

Direct observed dosing (TB)

Use more narrow spectrum antibiotics Use antimicrobial cocktails

Ecology of Antimicrobial Resistance

Antimicrobial peptides

› Broad spectrum antibiotics from plants and animals

Squalamine (sharks)

Protegrin (pigs)

Magainin (frogs)

Antisense agents

› Complementary DNA or peptide nucleic acids that binds to a pathogen's virulence

gene(s) and prevents transcription

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