Antimicrobial agents andantimicrobial resistance

BLS 206 Lecture

Hoza, A . S

Antimicrobial Resistance

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

Increase in MIC

Figure 20.20

Antibiotic Resistance

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

Mechanisms of Antibiotic Resistance

Antimicrobial Drug ResistancePrinciples and Definitions

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

Antibiotic Selection for Resistant Bacteria

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 resistanceG-neg bacteria are resistant to vancomycin (large molecule)

Tetracyclines are hydrophobic, G-neg bacilli are resistant

Acquired resistanceMutations (PBP)

Disseminated by plasmids and transposons

Spontaneous mutations

Types of resistance

Mechanisms of antibiotic resistance

1. Production of enzymesdestroying 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)

Bacteriophage virus, infecting bacteria (virus of bacteria)

Integron slice(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) antibioticsß-lactamases

Main mechanism of resistance in ß-lactam antibiotics

Penicillin-resistant S.aureus

Ampicillin-resistant E.coli

Production of enzymes modifying antibioticsAminoglycosides, chloramphenicol

(1) Mechanisms of resistance

Resistance mechanisms: inactivating enzymes (2)

Degrading enzymes will bind to the antibiotic and essentially degrade itor 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, AmpCESBL - 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

ExamplesTEM-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

Antimicrobial Drug ResistanceMechanisms

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

Antimicrobial Drug ResistanceMechanisms

Altered permeability› Altered efflux

tetracycline

Microbe Library

American Society for Microbiology

www.microbelibrary.org

Antimicrobial Drug ResistanceMechanisms

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 (macrolideresistantS.pneumoniae)

Mechanisms of resistance

Antimicrobial Drug ResistanceMechanisms

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 enterococci70% 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 drugresistant microorganisms

Interference with cell wallsynthesis

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 abbinds to PBP

2. Inhibition ofpeptidoglycansynthesis

3. Cell death

Structure of peptidoglycan

ß-lactams inhibit synthesis of crosslinks

Penicillins

Cephalosporins

Initially isolated formthe mould Cephalosporium

Compared with penicillins:More resistant to ß- lactamase hydrolysis

Wider antibacterial spectrum Improved PK-properties

Resistance to ß-lactamantibiotics

Resistance to ß-lactam antibiotics

Production of ß-lactamasesPenicillin-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

Glycopeptide resistance 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

Polypeptides 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 peptidoglycan precursors

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-positiveG-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

Aminoglycosides 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 actionAG 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 identifiedGenes encoding resistance located in plasmidsGene 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 syntheticNarrow 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 producingacetyltransferase 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 actionBlocking 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 BlockingTranslation 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

ResistanceIntrinsic 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

Antimicrobial Drug ResistanceMechanisms

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. SequencingUniversal 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 genes responsible for drug resistance development follwed by hybridization with oligonucleotide probes immobilized on membranes;

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

Molecular tests for the detection of resistance to RIF and INH

GenoType® MTBDRplus test procedure

Reaction zones of GenoType® MTBDRplus (examples)

What Factors Promote Antimicrobial Resistance?

Exposure to sub-optimal levels of antimicrobial

Exposure to microbes carrying resistance genes

Inappropriate Antimicrobial Use

Prescription not taken correctly

Antibiotics for viral infections

Antibiotics sold without medical supervision

Spread of resistant microbes in hospitals due to lack of hygiene

Inappropriate Antimicrobial Use

Lack of quality control in manufacture or outdated antimicrobial

Inadequate surveillance or defective susceptibility assays

Poverty or war

Use of antibiotics in foods

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

Consequences of Antimicrobial Resistance

Infections resistant to available antibiotics

Increased cost of treatment

MRSA “mer-sah”

Methicillin-Resistant Staphylococcus aureus

Most frequent nosocomial (hospital-acquired) pathogen

Usually resistant to several other antibiotics

Proposals to Combat Antimicrobial Resistance

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)

The Future of Chemotherapeutic Agents

Antisense agents› Complementary DNA or peptide nucleic

acids that binds to a pathogen's virulence gene(s) and prevents transcription

The Future of Chemotherapeutic Agents

Doctors are men who prescribe medicines of which they know little: to cure diseases of which they know less: in human being of who they know nothing’’

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