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ANTIMICROBIAL

RESISTANCE
9.21.12

Site of Action of antibiotics


Inhibition of nucleic acid synthesis (Rifampin; quinilones)
Inhibition of protein synthesis (Tetracyclines;

Chloramphenicol, macrolides, clindamycin,


aminoglycosides, linezolid)
Action on cell membrane (Polyenes; Polymyxin)
Interference with enzyme system (Trimethoprim,
Sulphamethoxazole)
Action on cell wall (Penicillin; cephalosporins, Vancomycin,
carbapenams)

Mechanisms of Drug Resistance


Change in drug target
Production of an enzyme that modifies or inactivates the

agent
Reduced accumulation of the agent
Limited uptake
Active Efflux

Loss of a pathway involved in drug activation

Mechanisms of Drug Resistance

Mechanisms of Drug Resistance

Mechanisms of Gram-Negative Bacterial


Resistance to Antibiotics
Antibiotic Class
Cephalosporins
-Lactamase
inhibitors
Carbapenems

Fluoroquinolone
s

Mechanism of Resistance
ESBLs

chromosomal

cephalosporinases

of -lactamases
new -lactamases resistant to inhibitors
chromosomal cephalosporinases
porin mutations
efflux pump overproduction (excluding
imipenem)
zinc metalloenzymes and other lactamases
alterations in DNA topoisomerase
efflux mechanisms
permeability changes
hyperproducers

Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

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Selection for antimicrobial-resistant


Strains

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Antimicrobial
Exposure

xx

Resistant Strains
Rare

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Resistant Strains
Dominant

Target Alterations
PBPs: in cell membrane
S. pneumoniae, MRSA
Intrinsic resistance, enterococci, gonococci, H. infl
D-Ala-D-Ala target: VRE
VanA, VanB, VanC, VanD

Alterations in ribosomes
Cell membrane changes

Protein Binding Proteins


Target for all B-lactams
found as both membrane-bound and cytoplasmic

proteins
all involved in the final stages of the synthesis of
peptidoglycan, which is the major component of
bacterial cell walls
More common R mechanism for gram positive
organisms

Gram neg access to PBP is limited by outer membrane

and thus other mechanisms supersede the binding to this


target

Enzyme Production
Aminoglycoside modifying enzymes
B-lactamases:
Four structural classes:
Class A: R of S aureus to penicillin, R of E coli to ampicillin and
cephalothin plasmid mediated
Class B: hydrolyze carbapenmens/pens/cephs -chromosomal
Class C: chromosomal, active against cephalosporins
Class D: plamid mediatated

ESBL: K. pneumoniae, E. coli : Derived from transfer of

chromosomal genes for inducible amp C onto plasmids

B-lactamase

B-lactame ring

Cefipime
Increased stability to B-lactamase
Increased penetration into gram-positive

Ceftriaxone

-Lactamases: Overview
Large, diverse family of enzymes
Widely dispersed in gram-positive (chromosoaml

and plasmid) and gram-negative pathogens


(plasmid)
Major mechanism of resistance to -lactams in
gram-negative pathogens
Wide range of activity: older enzymes hydrolyze
older drugs, new derivatives have evolved for
new drugs

ESBLs
AmpC -lactamases
carbapenemases

-Lactamases
Major groups for gram-neg
TEM-wide spread-plasmid and transposon
Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus

influenzae, and Neisseria gonorrhoeae


SHV-1
Klebsiella pneumoniae (chromosomal) and E. coli (plasmid)
Confer resistance to penicillins and first/second generation

cephalosporins
-lactamase

1960
TEM-2SHV
TEM-1

Extended spectrum-lactamase

1980s
Cefotaxime

TEM, SHV

CTX

ESBL-Mediated Resistance
Contain a number of mutations that allow them to

hydrolyze expanded-spectrum -lactam antibiotics

lactamase enzymes (TEM-1, TEM-2, SHV-1)

Derived from older antibiotic-hydrolyzing

a single amino acid substitution can give rise to new

ESBLs
Not as catalytically efficient
Inhibited by -lactamase inhibitors
Susceptible to cefoxitin and cefotetan in vitro only

10%40% of K pneumoniae, E coli express

ESBLs
Rupp ME et al. Drugs. 2003;63:353365.

CTM-X predominant mechanism

E. Coli predominant organism

Canton, Cur Opin in Micr 2006, Pages 466475

Coresistances among the Enterobacteriaceae isolates of the different ESBL types.

Morosini M et al. Antimicrob. Agents Chemother.


2006;50:2695-2699

Amp-C
Confer resistance cephamycins (cefotetan,

cefoxitin) and oxyimino- -lactams (cefotaxime,


ceftriaxone, ceftazidime)
Chromosomal in SPACE organisms and are
inducible
Poorly expressed in E. coli and is missing from

klebsiella and salmonella species


Plasmid mediated on other gram-neg, usually not

inducible
Not susceptible to inhibitors

AmpC- vs ESBL-Mediated
Resistance
Different phenotypic characteristics
AmpC type -lactamases typically encoded on

chromosome of gram-negative bacteria, can also


be found on plasmids
AmpC type -lactamases hydrolyze broad- and
extended-spectrum cephalosporins
ESBLsNOT AmpC -lactamasesare inhibited
by -lactamase inhibitors (eg, clavulanic acid)
AmpC production is less effective on cefipime so
best cephalosporin to test

New CLSI Laboratory Standards


Previously testing for ESBL was based on high MIC to

oxyimino-beta-lactam substrates (cetriaxone, cefotaxime,


cefipime, cetaz) and susceptibility to inhibitors followed by
a confirmatory test to detect the enzyme
Low sensitivity when mixed mechanisms at play, ie false positive

results, some attempts to overcome this with cloxacillin-containing


MullerHinton agar, which inhibits AmpC activity
When ESBL present susceptibility changed to resist for penicillins,
cephalosporins and monobactams

Current practice: MICs were changed


1-3 doubling dilutions lower
No need for confirmation of enzyme
No change in reporting

Epidemiology of Plasmid AmpC


Enzymes in the United States
Alvarez et al examined a sample of 752 resistant

K pneumoniae, K oxytoca, and E coli strains from 70 sites


in 25 US states
Plasmids encoding AmpC-type -lactamase were found

in
8.5% K pneumoniae samples
6.9% K oxytoca samples
4% E coli samples

Carbapenemases
beta-lactamases with versatile hydrolytic capacities.
Ability to hydrolyze penicillins, cephalosporins,

monobactams, and carbapenems.


2 major groups

Metallo-b-lactamases (MBLs)
Major R in pseudomonas, acinetobacter, and enterobacter
Confer High level of R
Serine b-lactamases
Oxacillinases or D b-lactamases (OxaA)
Not as Diverse
Found mostly in acinetobacter
Confer only low level of hydrolytic activity therfore another R is necessary to
raise MIC

Class A carbapenemases
Found in pseudomonas and enterobacter, but predominant type is found on a
plasmid in Klebsiella

Mechanisms of Bacterial
Resistance to Fluoroquinolones
Mutations in DNA gyrase and topoisomerase
Overexpression of efflux pump system
Bacterial membrane permeability changes

Mechanisms of Antibiotic Resistance in


Nonfermenters
P aeruginosa and Acinetobacter often multidrug

resistant1
Mechanisms of resistance include1,2
production of ESBLs or AmpC -lactamases
increased efflux of antibiotic agent
decreased outer membrane permeability
DNA gyrase mutations
aminoglycoside modifying enzymes

Carbapenems: Resistance Issues


Mechanisms of resistance to carbapenems in

P aeruginosa involve

loss of OprD protein (initially called D2 porin)


overproduction of efflux pump system

(MexA-MexB-OprM)
upregulation of other efflux system may be involved (crossresistance to fluoroquinolones)

Resistance to meropenem depends on both


Resistance to imipenem mainly mediated

through loss of OprD

Carbapenems: Resistance
Issues
Carbapenem nucleus

Ertapenem

Imipenem

Mutated or
missing
D2 porin

D2 Porin (OprD)

Outer
membrane
Periplasm

Cytoplasmic
membrane

Penicillin-binding
proteins (PBPs)
PBP
1

Courtesy of John Quinn, MD.

PBP
2

PBP
3

PBP
4

PBP
5

Mechanisms of Carbapenem
Resistance: Impermeability
OprD forms narrow transmembrane channels that

are normally accessible only to carbapenems, not


to other -lactams
Loss of OprD porin is associated with decreased
permeability of carbapenems and increased
carbapenem MICs, whereas other -lactams
remain active

Mechanisms of Carbapenem Resistance:


Efflux Systems in P aeruginosa
Upregulation of MexAB-OprM efflux system
associated with increased MICs of meropenem, not
imipenem
Coregulation of MexE-MexF-OprN efflux system

with OprD porin in P aeruginosa


upregulation of efflux associated with OprD
associated with increased MICs of fluoroquinolones as

well as carbapenems
mechanism sometimes selected by fluoroquinolones,
rarely by carbapenems

MRSA
Methicillin resistance is acquired via Mec A
mobile chromosomal element called staphylococcal cassette
chromosome (SCCmec)
SCCmec types I, II, and III and are multidrug resistant-large cassettes
Health-care associated
SCCmec type IV and type V not multidrug resistant
Community associated

MecA
Encodes penicillin binding protein (PBP) 2a
Weak affinity for methicillin and all beta-lactams
Substitutes for the usual PBP 1-3 that have a high affinity for betalactams
Speculation of origination from CoNS

S. Pneumoniae
Pencillin
Decreased affinity to PBP
Can be overcome with high dose

Macrolides
Genetic changes to binding target on ribosome-high

level can not be overcome =erm(B)


Efflux pump-lower level-may be overcome =mef (A)
Clindamycin
Ribosomal methylation changing target erm(B)

S. pneumoniae
Fluoroquinilones
Bind to either gyrase or topoisomerase or both
Resistance from mutations in gyrA or parC
reduce binding of the drug to the site of activity
Mutations are step wise
One mutation and R to cipro and levo
More than one needed for gemi and moxi

Tetracyclines
Proteins are produced that package the drug into vessicles which
are extruded from the cell

Enterococcus
Intrinsic (chromosomal, naturally occurring) resistance to
B-lactam
10 to 1000 times more drug to inhibit an average Enterococcus than an

average Streptococcus
Due to penicillinase production and PBP5 production
Aminogylcosides
Low level to streptocmycin and gentimicin
Synergism causes cell wall agent to become bactericidal
High level to tobramycin

Enterococcus-Intrinsic
Clindamycin-gene encoding efflux pump
TMP-SXZ In vitro appears susceptible but in vitro is resistant
Can utilize preformed folic acid

Vancomycin at low levels in some strains

Enterococcus
Genetic transfer to acquire new resistance
One mechanism, involving pheromone-responsive
plasmids, causes plasmid transfer between E. faecalis
isolates at a very high frequency .
Another mechanism involves plasmids that can transfer
among a broad range of species and genera, although
usually at a moderately low frequency .
A third mechanism (conjugative transposition) involves
transfer of specialized transposons at low frequency but
to a very broad range of different kinds of bacteria .
Conjugative transposons are relatively nonselective in
their host range and are one of the few types of elements
known to have crossed the gram-positive/gram-negative
barrier in naturally occurring clinical isolates and to then
cause resistance in these various hosts

Enterococcus
Acquired
High level resistance to amnioglycosides
Loose synergy ability as well

High level vancomycin resistance


Van gene clusters on transposons or plasmids
Very old, probably initially resulted from pressor from natural glyocpeptides
Van A is the most common and confers highest level of resistance

Variable level to linezolid


Depends on the number of mutations in the 23S rRNA