mechanisms of resistance

Antibiotic Resistance:

An Introduction to Mechanisms

T. Mazzulli, MD, FRCPC, FACP

Microbiologist & Infectious Disease Consultant

Mount Sinai Hospital/University Health Network

Antibiotic

Mechanisms of Action and Resistance

Objectives1. Understand the factors affecting the interaction

between antimicrobial agents and infecting organisms

2. Recognize and explain (with examples) the phenotypic and genotypic mechanisms of resistance

3. Understand some key concepts related to mechanism of action and resistance of common antimicrobial agents

Mechanisms of Antimicrobial Resistance

1. Phenotypic

2. Genotypic

Phenotypic Mechanisms of Antimicrobial

Resistance

Mechanism Example

Enzymatic inactivation/inactivation of the drug Impermeable cell wall Efflux Target modification Target by-passing

Beta-lactamases Aminoglycoside-modifying enzymes Glycopeptides vs. Gram-negatives Imipenem-resistance in P. aeruginosa Most tetracycline resistance in Gram-negatives Multi-drug efflux in P. aeruginosa Quinolone resistance in S. aureus Glycopeptide resistance in enterococci Penicillin-resistance in pneumococci Methicillin-resistance in S. aureus Most anti-folate resistance

Phenotypic Mechanisms of Antimicrobial

Resistance

Mechanism Example

Protection of target site Overproduction of target Bind-up antibiotic

Tetracyclines Sulfonamides, Trimethoprim, Glycopeptides Glycopeptides

Genotypic Mechanisms of Antimicrobial

Resistance

2 key mechanisms:

1. Intrinsic (Primary)

2. Acquired (Secondary)


Resistance

1. Intrinsic Resistance:– Usually related to structural features

e.g. permeability of cell wall or target modification

– Chromosomally mediated

e.g. Pseudomonas aeruginosa, S. maltophilia, Enterococci, others


Resistance

2. Acquired Resistance:

i. Point Mutation's - random (10-6 - 10-8)

- chromosomal

ii. Acquisition of resistance genes

[Plasmids, Transposons, other DNA]

- Transduction

- Transformation

- Conjugation

Transduction

Transformation

Conjugation

Selected Issues With Acquired Antimicrobial

Resistance

Antibiotic Class Mechanism of Resistance

Cephalosporins Vancomycin/Teicoplanin

Quinolones Macrolides Aminoglycosides

Penicillin / -lactam

Extended-Spectrum -lactamases (ESBL) Chromosomal Cephalosporinases Modified cell wall precursors with decreased affinity for vancomycin (e.g. VRE)

Alterations in DNA topoisomerase, efflux mechanisms, permeability changes Methylation of bacterial ribosome (MLS phenotype); Efflux (M phenotype) Aminoglycoside-modifying enzymes

Altered Penicillin-binding proteins (eg. MRSA, PRSP)

Resistance Due to Antibiotic Selection

Spontaneous

mutation occurs

in the absence

of drug

selection in a

sensitive

population

Drug

treatment

Mutant is selected

for by drug

treatment as

sensitive strains

die off

Resistant

clone grows

within what

used to be a

sensitive

population

Sensitive

bacteria

Resistant

bacteria

Resistant clone

becomes dominant

(may be multi-drug

resistant)

Dissemination of Resistant Bacteria

Resistant clone

spreads to other

patients &

contacts

Resistant clone

spreads to the

environment

Resistant clone results in

failure of therapy or re-

infection/relapse in the same

patient

Controlling Antimicrobial Resistance

• Reducing colonization and infection

• Reducing volume of antimicrobial use

• When decision made to treat

– Use right drug

– Right dose

– Right duration

Controlling Antimicrobial Resistance

Antimicrobial Stewardship Infection Control

Mechanisms of Antibiotic Resistance

1. Enzymatic inactivation:

• Beta-lactamases

• Aminoglycoside-modifying enzymes

Ambler Classification of Beta-Lactamases

Class Active Site Enzyme Type Substrates

A Serine Penicillinases

Broad Spectrum Pen. (all), Narrow Ceph.

ESBL Above plus 3rd Gen. Ceph, Aztreonam

Carbapen’ase Above plus Cephamycins,

Carbapenems

B Met-B-Lac (Zn2+) Carbapen’nase Above plus Cephamycins,

Carbapenems

C Serine Cephalosp’nase Extend. Spectrum + Cephamycins

D Serine Oxacillinases

Broad Spectrum Pen. (all), Narrow Ceph.

Extend. Spectrum Broad Spectrum + Monobactam

Carbapen’nase Extended Spectrum + Cephamycins,

Carbapenems

Jacoby GA et al. NEJM 2005;352

Bush-Jacoby-Medeiros Functional Classification of Beta-Lactamases

Group Enzyme Type Inhibited by Clavulanate Molecular Class

1 Cephalosporinase No C

2a Penicillinases Yes A

2b Broad Spectrum Yes A

2be Extended Spectrum Yes A

2br Inhibitor-Resistant Diminished A

2c Carbenicillinases Yes A

2d Cloxacillinase Yes D or A

2e Cephalosporinase Yes A

2f Carbapenemase Yes A

3 Carbapenemase No B

4 Penicillinase No A

Jacoby GA et al. NEJM 2005;352

Increase in numbers of Group 1, 2 and 3 β-

lactamases from 1970 to 2009

Group 1/class C cephalosporinases

Group 2/class A and class D β-lactamases

Group 3/class B metallo-

β-lactamases

Bush K and Jacoby G. AAC 2010

Group 1 (Class C) - Amp C -lactamases

• Cephalosporinases; chromosomally mediated; Inducible

• Typically found in Enterobacter, C. freundii, certain

indole-positive Proteae, Serratia, P. aeruginosa (not

Klebsiella or Salmonella spp.) - “SPICE” organisms

• Hydrolyzes penicillins, cephlalosporins, monobactams,

and cephamycins

• Resistant to -lactamase inhibitors

• Only carbapenems and cefepime are active (but

carbapenems and cefoxitins are strong inducers)

• May move onto plasmids and become constitutive


• In E. coli, constitutive over expression of AmpC

β-lactamases can occur because of:

– mutations in the promoter and/or attenuator region

(AmpC hyperproducers)

– the acquisition of a transferable ampC gene on a

plasmid or other transferable elements (plasmid-

mediated AmpC β-lactamases)


Laboratory implications:

• MICs > 8 ug/ml

• Easily identified in lab if expressed

– No “inducibility” test

Group 2 (Class A) -lactamases

• Dozens of enzymes; may be plasmid (constitutive)

or chromosomal (inducible)

• Non-essential enzymes

1. Broad Spectrum:

– PC1 in Staphylococcus aureus:

• Due to PC1

• Prevalence >95% worldwide

• Beta-lactamase stable penicillins (e.g. cloxacillin, nafcillin) and

beta-lactamase inhibitors are effective

• Hyperproduction results in “Borderline Oxacillin resistant S.

aureus” (BORSA) with MIC=4ug/ml; do not have the same

infection control implications as MRSA


1. Broad Spectrum:

– H. influenzae:

• Due to TEM-1 (90%) and ROB-1 (10%) beta-

lactamase

• Prevalence now 25-40% worldwide

• Initially more prevalent in serotype b strains

– M. catarrhalis:

• prevalence now >95% worldwide

• BRO-1 (predominates) and BRO-2 enzymes differing

by one amino acid

• Readily transferred by conjugation


1. Broad Spectrum:

– Most common -lactamases found in E. coli and K.

pnuemoniae are plasmid mediated TEM-1, TEM-2,

SHV-1

• Responsible for ampicillin resistance in E. coli &

ampicillin-cephalothin resistance in K.

pnuemoniae

• Susceptible to -lactamase inhibitors


2. Extended Spectrum (ESBLs):

– >20 derivatives of TEM; 6 derivatives of SHV; others

• Rapidly increasing are CTX-M -lactamases which were acquired

via plasmids from the chromosomal Amp C enzymes of Kluyvera

spp. (environmental gram negatives)

– Amino acid substitutions in area of -lactamases capable of

accommodating bulkier side chains of newer

cephalosporins & aztreonam

– Resistant to all penicillins, most inhibitor/drug combinations,

cephalosporins and aztreonam

– Carbapenems and cephamycins (cefotetan, cefoxitin) are

active in vitro

– Multi-resistant to other classes of drugs


2. Extended Spectrum (ESBLs):

– Predominant bugs: E. coli & K. pnuemoniae

– Occasionally found in K. oxytoca, K. ozaenae,

Serratia marcescens, Enterobacter, Salmonella,

Proteus, Citrobacter, Morganella morganii

Extended Spectrum Beta-lactamases (ESBLs)

Laboratory implications:

• Modest increase in MICs (1 to 8 ug/ml)

• Difficulty in detecting them if only screening

with a single 3rd generation cephalosporin

(e.g. cefotaxime)

• New CLSI guidelines

Enterobacteriaceae: Breakpoints revised

Agent

CLSI 2009 CLSI 2010

S I R S I R

Cefazolin ≤8 16 ≥32 ≤1 2 ≥4

Cefotaxime ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftriaxone ≤8 16-32 ≥64 ≤1 2 ≥4

Ceftazidime ≤8 16 ≥32 ≤4 8 ≥16

Aztreonam ≤8 16 ≥32 ≤4 8 ≥16

Cefipime ≤8 16 ≥32 ≤8 16 ≥32


3. Carbapenemases:

– KPC-1, KPC-2, KPC-3 in K. pneumoniae are most

prevalent:

• Now reported in many other gram negatives – E. coli,

Citrobacter, Enterobacter, Salmonella, Serratia, P. aeruginosa

• KPC enzymes are transferable on plasmids

– Hydrolyze carbapenems, broad-spectrum penicillins,

oxymino-cephalosporins, and cephamycins

– Not inhibited by -lactamase inhibitor combinations

– Subgroup 3b contains smaller group of MBLs that

preferentially hydrolyze carbapenems

• IMP & VIM enzymes most frequently in non-fermentative

bacteria but also in Enterobacteriaceae

Implications of B-lactamases in Gram Negatives

• ESBLs - consider all pencillins, cephalosporins & aztreonam as resistant; ? Beta-lactamase inhibitors

• AmpC - Enterobacter, Citrobacter & Serratia spp. -may develop resistance to 3rd Gen. Ceph within 3 to 4 days of therapy

• Salmonella & Shigella spp. should always be considered resistant to 1st & 2nd Gen. Ceph.

• For enterobacteriaceae: Cephalothin R = Cephalexin R but not Cefazolin R

Implications of B-lactamases in Gram Negatives

• 1st Gen. Ceph. - R: Citrobacter, Enterobacter,

Morganella, Providencia, Serratia, Proteus vulgaris,

Yersinia

• Ampicillin - R: Above plus Klebsiella

• 2nd Gen. Ceph. - R: Citrobacter, Enterobacter,

Serratia, (P. vulgaris - cefuroxime)

• Amox/Clav. - R: Citrobacter, Enterobacter, Serratia


1. Enzymatic inactivation:

• Beta-lactamases

• Aminoglycoside-modifying enzymes

Enterococcal Aminoglycoside-Modifying Enzymes

Organism Enzyme STREP GENT TOBRA AMIK

E. faecalis & faecium

6-AAD

+

-

-

-

3’-APH - - - +

2”-APH/ 6’-AAC

- + + +

E. faecium 6’-AAC* - - + +

4”-AAD - - + +

+ = synergy NOT achievable; - = synergy achievable with cell wall agent

*Intrinsic, chromosomally-mediated

Aminoglycoside


2. Target modification:

• Penicillin resistance in pneumococci (PRSP)

• Vancomycin resistant enterococci (VRE)

3. Target by-passing:

• Methicillin-resistance in S. aureus (MRSA)

-lactam Antimicrobials

Mechanism of Action:

• Target: penicillin binding proteins (PBP)

• PBPs are essential enzymes

(carboxypeptidases, transpeptidases &

transglycosylases) for building the bacterial

cell wall

• Multiple PBPs in different organisms

Glycopeptide Antimicrobials


– Exact mechanism of action not known

– Binds to terminal D-ala-D-ala residues of

cell wall components and prevents

incorporation of the subunit into the

growing peptidoglycan

-lactams Antimicrobials

2. Altered Target (Remodeling):

• S. pneumoniae: slow remodeling of PBPs (3 of

the 6 PBPs are altered - 1a, 2x, & 2b)

• Due to transformation of PBP genes via

scavenging of genetic material– Gradual increase in MICs (<0.06 to 0.5 / 1.0)

» 3 of 6 altered PBPs for Pen Resistance

» 2 of 6 altered PBPs for Ceph Resistance

• High Level resistance when MIC >4 ug/ml

• Concomitant resistance to other unrelated

classes of antibiotics (~10 to 15% are MDR)

Lab Implication of Penicillin Resistance

in S. pneumoniae

Source of isolate and

mode of therapy

Susceptibility

category

(MICs: µg/ml)

S I R

Meningitis isolates ≤0.06 .12 -1 ≥2

Non-meningitis isolates, oral ≤0.06 .12 -1 ≥2

Non-meningitis isolates,

parenteral

2 4 8

CLSI 2008. Performance Standards for Antimicrobial Susceptibility Testing;

Eighteenth Informational Supplement (M100-S18)

-lactams Antimicrobials

3. Target By-passing (Novel PBP):

• S. aureus- synthesis of a novel PBP 2a

(capable of continuing cell wall synthesis)

MRSA (encoded for by chromosomal mecA

gene)

• Hospital MRSA (multi-drug resistant) vs

Community MRSA (less drug resistant)

Classification of MRSA

US PFGE Canadian strain SCCmec Type PVL

USA 100 2 II -

USA 200 3 – 6 II -

USA 300 10 IVa +

USA 400 7 IVa +

USA 500 5 – 9 IV -

USA 600 1 II -

USA 700 - IV -

USA 800 2 IV -

USA 1000 - IV +

USA 1100 - IV +


4. Target modification and efflux:

• MLS antibiotics

MLS Antibiotics

• Macrolides - Erythromycin

- Azithromycin

- Clarithromycin

• Ketolides - Telithromycin

• Lincosamides - Clindamycin

• Streptogramins - Quinipristin /

Dalfopristin

Mechanism of Action & Resistance

• Interact with 50S ribosomal subunit (mainly with

23sRNA)

– Inhibition of bacterial protein synthesis

• Macrolides bind strongly to Domain V and weakly to

Domain II, whereas ketolides bind strongly to both

domains

• Do not induce target resistance nor are they

affected when it has been induced by others;

(Telithromycin not affected by efflux mechanism)

Mechanisms of Macrolide Action & Resistance

Cytoplasm

Ribosomes

50

30

50

30

50

30

Bacteria alter macrolide binding site

(ermAM gene, MLSB phenotype)

Macrolide unable to block protein synthesis

Macrolide

Bacteria activate efflux pumps

(mefE gene, M phenotype)

Macrolide excreted from cell

MLS Antibiotics – Mechanisms of Resistance

1. Target modification (erm genes):

• Inducible - S. pneumo - all MLS

- S. aureus - only M

• Constitutive

• Acquisition of a gene; one step

• MIC increases from < 0.5 to > 8.0

mg/L

MLS Antibiotics – Mechanism of Resistance

2. Efflux pump (mef genes):

• S. aureus - MS phenotype

• S. pneumo - MLS phenotype

• Rare in S. aureus

3. Inactivation:

• Rare in S. aureus

• Not seen in S. pneumoniae

0

10

20

30

40

50

No

. o

f S

train

s

Erythromycin MIC (g/mL)

mefE +ermB

ermB

mefE

None

Correlation Between Erythromycin

MIC and Resistance Mechanisms

MLS Resistance in S. pneumoniae

Cross-Resistance

Mechanism % of Macrolide

Resistance

Clindamycin Streptogramin

Altered target Efflux pump

45

55

Yes

No

Yes

No

MLS Resistance in S. aureus

Cross-Resistance

Mechanism % of

Macrolide

Resistance

Clindamycin Streptogramin

Altered target

Efflux pump

>98

Rare

Yes

No

Yes

Yes

Clinical Implications of MLS Resistance

• Macrolide Resistant / Clindamycin Sensitive:

• S. aureus - do not use clindamycin

(inducible resistance)

• S. pneumoniae - could use clindamycin

(efflux pump - no cross-resistance)


5. Target Modification:

– Fluoroquinolone resistance in S. pneumoniae

Fluoroquinolones


• Bind to 2 essential enzymes

• DNA gyrase (topoismerase II)

• Topoisomerase IV

• Results in termination of nucleic acid synthesis

and replication

• Bactericidal - kill both multiplying and resting

bacteria

Fluoroquinolones


• DNA gyrase (gyr A / gyr B)

• 10 target in gram neg

• 20 target in gram pos

• Topoisomerase IV (par C / par E)

• 10 target in gram pos

• 20 target in gram neg

Topoisomerases

• Enzymes which alter the number of times one

single strand of DNA duplex winds around its

complimentary strand

• Essential enzymes

• DNA replication, recombination, transcription

• Role in partitioning replicated chromosomes

Fluoroquinolones

Mechanism of Resistance:

1. Decreased permeability - low level resistance

2. Energy dependent efflux - low level resistance

3. Target modification - high level resistance

– Point mutations of gyr A & par C

– No plasmid (transferable) resistance until recently

Fluoroquinolones

Mechanism of Resistance:

• 1st / 2nd generation agents

• single mutations required for clinically

relevant resistance

• 3rd / 4th generation agents

• two mutations required for clinically

relevant resistance

Vancomycin Resistance

• Enterococcus

• S. aureus

Enterococci - Instrinsic Resistance (“Low level”)

• Penicillins and glycopeptides inhibit but do

not kill (MICs of 1 - 2 mg/L)

• Cephalosporins - uniformly resistant

• Vancomycin (Van C) - all E. flavescens, E.

casseliflavus, E. gallinarum; not transferable

• Aminoglycosides - poor drug uptake (MIC = 8

-256 mg/L)

Enterococci - Acquired Resistance (“High Level”)

• Beta-lactams - altered PBPs; MIC >128 mg/L

- beta-lactamase; plasmid, similar to

S. aureus (rare)

• Aminoglycosides - plasmid-mediated inactivating

enzymes; MIC >500 mg/L

• Vancomycin (& Teicoplanin) - Van A (transposon)

& Van B (chromosomal but

transferable by conjugation)

Implications of Enterococcal Resistance

• All cephalosporins are resistant

• High level aminoglycoside R - no synergy

• Quinapristin/dalfopristin (Synercid) - active against

E. faecium but not E. faecalis

• Linezolid active against both including VRE

• Nitrofurantoin for VRE in urine

• Altered PBPs also resistant to carbapenems

Classes of Vancomycin Resistance in

Enterococcus spp. & S. aureus

Vancomycin Teicoplanin

Class MIC ug/ml MIC ug/ml Inducible Location Species

A 64 to >500 (R) >32 (R) Yes P, C E. faecalis

E. faecium S. aureus

B >16-1024 (I,R)

<8 (S) Yes P, C E. faecalis

E. faecium

C 2 - 32 (I,R) 0.5 – 2 (S) No/Yes C E. galinarum

E. casseliflav

D 64 – 128 (R) 4 – 64 (S,I,R)

No C E. faecium

E 16 (I) 0.5 (S) Yes C E. faecalis

G 12 – 16 (I) 0.5 (S) ? C E. faecalis

Vancomycin Resistance

• Due to target alteration:

– D-ala-D-lac: Van A, B, D

– D-ala-D-ser: Van C, E, G

VRSA: Vancomycin-Resistant S. aureus

• First identified in US 2002

• High-level resistance (MIC >16 µg/mL)

• vanA and associated genes from VRE

Vancomycin and S. aureus Resistance

1. Vancomycin Intermediate S. aureus (VISA) &

Heteroresistant-VISA (hVISA):

• 1st case of VISA reported in Japan in 1997

• hVISA is a precursor of VISA:

– Heterogeneous pop’n of S. aureus with MIC of <2 ug/ml but

with non-susceptible subpopulations (MIC >4 ug/ml)

– 2.16% of MRSA and 0.05% MSSA strains (range 0% to

74%)

2. Vancomycin MIC Creep:

• Pop’n shift in MICs over time

S. aureus with Reduced Susceptibility to

Vancomycin

VISA: vancomycin-intermediate S. aureus

• Japan 1997 (Mu50)

• MIC 8 to 16 µg/mL

• Linked to cell wall thickening

hVISA: vancomycin heteroresistant

• Japan 1997 (Mu3)

• Subpopulations of cells in intermediate range

• MIC ≥4 µg/mL

hVISA and VISA: Mechanism of Resistance

• Overexpression of the vraSRtwo-component system

• Thickened cell walls contain an increased number of cell wall D-Ala-D-Ala residues

• Creates a reservoir that effectively sequesters vancomycin and prevents it from reaching the lethal target sites

http://www.journals.uchicago.edu.myaccess.library.utoronto.ca/action/showFullPopup?doi=10.1086/533592&id=_e5

Vancomycin Breakpoints for S. aureus

• April 2008, FDA set new breakpoints for Vancomycin and are

consistent with those set by the CLSI in 2006

– Based on increasing reports of treatment failures, greater ability to detect

heterogeneously resistant isolates and reports of upward trend of vancomycin

MIC values in S. aureus

Drug

S, I, R Breakpoints (µg/ml)

CLSI (2006) FDA (Pre-2008)

Oxacillin ≤2, -, ≥4 ≤2, -, ≥4

Vancomycin ≤2, 4-8, ≥16 ≤4, 8-16, ≥32

Vancomycin Therapeutic Guidelines

• Vancomycin displays concentration-independent activity against S. aureus,

• AUC/MIC of 400 is the target

• A loading dose of 25–30 mg/kg should be considered

• Trough serum vancomycin concentrations

• Should be obtained just before the 4th dose

• 15–20 mg/L are recommended

• Dosages of 15–20 mg/kg q8–12h are required for most patients with normal renal function if MIC is <1 µg/mL

• If MIC >1 µg/mL, alternative agent recommended

Antibiotics - Summary

• Understanding mechanism of action / resistance should

allow selection of appropriate empiric therapy

• Pharmacokinetic / Pharmacodynamic properties important in

understanding how antibiotics work and implications of

resistance

• Low level resistance may be overcome with higher doses

(e.g. PRSP) or combination therapy (e.g. amp + gent)

• Co-resistance may or may not be predictable

• Susceptibility does not predict clinical success, but

resistance may increase likelihood of failure

Thank you for your attention!

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