Transcript Step-wise resistance due to low

Mechanisms of Resistance


Antibiotics exert selective pressure that favors
emergence of resistant organisms
Bacteria employ several biochemical strategies
to become resistant
Decreased
permeability
Inactivation
Efflux
X
Altered
target
1
Genetic Basis of Resistance

Spontaneous mutations in endogenous genes
– Structural genes: expanded spectrum of enzymatic
activity, target site modification, transport defect
– Regulatory genes: increased expression

Acquisition of exogenous sequences
– Usually genes that encode inactivating enzymes or
modified targets, regulatory genes
– Mechanisms of DNA transfer: conjugation (cell-cell
contact); transformation (uptake of DNA in solution);
transduction (transfer of DNA in bacteriophages)

Expression of resistance genes
– Reversible induction/repression systems can affect
resistance phenotypes
2
Spread of Resistance Genes
Conjugation
R
R
S
R
Transformation
S
R
R
3
Major Classes of Antibiotics
Mechanism of action
Major resistance
mechanisms
Inactivate PBPs
(peptidoglycan
synthesis)
• Beta-lactamases
Bind to precursor of
peptidoglycan
• Modification
Aminoglycosides
Inhibit protein synthesis
(bind to 30S subunit)
• Modifying
Macrolides
Inhibit protein synthesis
(bind to 50S subunit)
• Methylation
Quinolones
Inhibit topoisomerases
(DNA synthesis)
• Altered
Beta-lactams
Glycopeptides
• Low
affinity PBPs
• Decreased transport
of
precursor
enzymes
(add adenyl or PO4)
of rRNA
• Efflux pumps
target enzyme
• Efflux pumps
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Antibiotic Susceptibility Tests

Minimal inhibitory concentration (MIC)
– Reference method. Add standard inoculum to dilutions of
antibiotic. Incubate overnight. MIC is lowest concentration that
inhibits growth (can also be performed by agar dilution).
– Interpretation (S or R) is based on achievable drug levels
104 cfu
mg/ml
4
2
1
0.5
0.25
0.12
0
5
Antibiotic Susceptibility Tests

Kirby-Bauer agar disk diffusion
– Paper disk containing antibiotic
is placed on lawn of bacteria,
then incubated overnight.
Diameter of zone of inhibition is
inversely related to MIC (used to
establish interpretive
breakpoints).
– Standardized for commonly
isolated, rapidly growing
organisms.
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Antibiotic Susceptibility Tests

E-test
– Strips containing a gradient of
antibiotic are placed on lawn of
bacteria and incubated overnight.
MIC is determined at point where
zone of inhibition intersects scale
on strip.
– Combines ease of KB with an MIC
method. Particularly useful for S.
pneumoniae.
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b-lactam Antibiotics
– Substrate analogs of D-Ala-D-Ala
– Covalently bind to PBPs, inhibit final step of
peptidoglycan synthesis
S
S
N
O
N
Cephalosporins

1st gen: GPC, some GNR

2nd gen: some GNR
+anaerobes

3rd gen: many GNR, GPC
O
Penicillins
N
N
O
Carbapenems
O
Monobactams
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Structure of Peptidoglycan
cytoplasm
NAG-NAM-NAG-NAM
|
NAG-NAM-NAG-NAM
L-Ala
|
|
L-Ala
D|
Glu
-(AA)n-NH2 D-Glu
|
|
L-diA
L-diA-(AA)n|
| NH2
D-Ala
D-Ala
|
|
D-Ala
D-Ala
Transpeptidation
reaction
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Penicillin-Binding Proteins (PBPs)

Membrane bound enzymes
– Catalyze final steps of peptidoglycan synthesis
(transglycosylation and transpeptidation)
– Multiple essential PBPs (4-5) - involved in cell
elongation, determination of cell shape, and cell
division; essential for cell viability

b-lactams acylate active site serine of PBPs,
inhibit transpeptidation
– Activity determined by affinity for PBPs, stability
against b-lactamases, and permeability
– Autolysins contribute to bactericidal activity
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Penicillin-Resistant S. pneumoniae (PRSP)

S. pneumoniae interpretative breakpoints
– penicillin susceptible (MIC  0.0625 µg/ml),
intermediate (0.125 -1.0), resistant ( 2.0).

High-level penicillin resistance has risen
rapidly in US (0.01% in 1987 to 3% in 1994)
– 20-30% of isolates may be non-susceptible (I or R).
– High-level PRSP may exhibit cross-resistance to
3rd generation cephalosporins
– Serious problem when infection occurs at body
sites where antibiotic availability is limited.

PRSP may be multi-resistant (macrolides,
TMP/SXT); strains can spread widely
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Mosaic PBP Genes in PRSP

Resistance is due to alterations in endogenous
PBPs
– Resistant PBP genes exhibit 20-30% divergence
from sensitive isolates (Science 1994;264:388-393)
– DNA from related streptococci taken up and
incorporated into S. pneumoniae genes
S SXN
PBP 2B
Czechoslovakia (1987)
South Africa (1978)
USA (1983)
= pen-sensitive S. pneumoniae
= Streptococcus ?
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International Spread of PRSP

Multiresistant PRSP in Iceland (JID 1993;168:158-63)
– First isolate in 12/88; 17% PRSP in 1992.
• Almost 70% of PRSP were serotype 6B; resistant to tet,
chloram, erythro, and TMP/SXT; similar or identical
molecular markers.
• Icelandic PRSP identical to multiresistant 6B clone
endemic in Spain (popular vacation site).

Possible factors responsible for rapid spread
– b-lactam use in Iceland low, but high use of
TMP/SXT, tet, etc may have selected for
multiresistant clone.
– 57% of population lives in Reykjavik/suburbs, almost
80% of children age 2-6 attend day-care centers.
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b-lactam resistance in Staph. aureus

>90% of strains produce b-lactamase
– plasmid encoded, confers resistance to penicillin,
ampicillin
– these strains are susceptible to penicillinaseresistant penicillins (e.g. methicillin), 1st generation
cephalosporins, and b-lactam/b-lactamase inhibitor
combinations

At many large medical centers, approx 30% of
S. aureus are resistant to methicillin and other
b-lactams
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Methicillin-resistant S. aureus (MRSA)

MRSA contain novel PBP2a, substitutes for
native PBPs; low affinity for all b-lactams
– MRSA chromosome contains ~ 50kb mec region
not present in MSSA. Acquired from coag-neg
Staph spp.
– PBP2a is encoded by mecA gene; expression
controlled by mecI, mecR1 and other factors.


Most MRSA are also resistant to macrolides
and fluoroquinolones; remain susceptible to
vancomycin.
Major nosocomial pathogen; primarily spread
on hands of healthcare workers.
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Enterococci and b-lactams

Intrinsically less susceptible to b-lactams
– PenG/Amp MICs 10-fold higher than other
streptococci, not bactericidal
• PenG/Amp + gent (bactericidal) for endocarditis
• Ampicillin alone effective for UTI
– Cephalosporins not active; increase risk of
enterococcal infection

Acquired resistance is a new problem
– High-level Amp resistance (altered PBPs in E.
faecium); [b-lactamase still rare]
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Vancomycin-resistant Enterococci


Since 1989, a rapid increase in the incidence
of infection and colonization with vancomycinresistant enterococci (VRE) has been reported
by U.S. hospitals (MMWR Vol. 44 / No. RR-12)
This poses important problems, including:
– Lack of available antimicrobial therapy for VRE
infections because most VRE are also resistant to
drugs previously used to treat such infections (e.g.
aminoglycosides and ampicillin).
– Possibility that vancomycin-resistance genes
present in VRE can be transferred to other grampositive bacteria (e.g. Staph. aureus )
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Vancomycin

Member of glycopeptide family
– Binds to D-Ala-D-Ala in peptidoglycan precursors
– Prevents transglycosylation and transpeptidation
– Resistance to b-lactams does not confer crossresistance to vancomycin

Only active against Gram-positives
– Cannot cross outer membrane of Gram-negatives
– Primarily used for MRSA, MRSE infections; pts with
penicillin allergy; severe C. difficile disease.
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Mechanism of Action of Vancomycin
G
M
Vancomycin
G
M
G
M
G
M
Vancomycin binds to
D-Ala-D-Ala; prevents
transglycosylation
and transpeptidation
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Mechanism of VRE


Acquired high-level resistance
– E. faecium and E. faecalis containing vanA or
vanB gene clusters produce modified peptidoglycan
containing D-Ala-D-lactate; does not bind
vancomycin (MIC = 32 - >256
– Resistance genes are on mobile elements, have
spread widely since 1st reports in late 80’s; major
focus of infection control
– Multiresistant E. faecium (vancomycin, high-level
ampicillin, high-level aminoglycoside) poses
therapeutic challenge
Other enterococci contain vanC; low-level, nontransferable resistance; strains have low pathogenicity
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Vancomycin
Resistance
pyruvate
G
M
G
M
vanH
vanA
+
D-Lac D-Ala
ddl
+
vanX
Vancomycin does not
bind to modified
peptidoglycan
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Epidemiology of VRE

Risk factors for colonization/infection in USA
– Severe underlying disease (malignancy, ICU, long
hosp); antibiotics (vancomycin, 3rd gen cephs)

Reservoirs, routes of dissemination not fully
understood
– VRE strains can be distinguished by molecular
typing (PFGE)
– Multiple patterns are seen in some institutions
(endogenous infection from intestinal source?)
– Clonal outbreaks are seen in others (transmission
by HCWs?, fomites?)
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VRSA - An emerging Problem

Several reports of S. aureus with reduced
susceptibility to vancomycin since 1997
– Japan and U.S. (Michigan, NJ, NY, Illinois)
– Vancomycin MIC = 8 mg/ml
– Isolates obtained from patients with chronic MRSA
infection


No evidence of vanA or vanB
Decreased susceptibility due to increased
levels of peptidoglycan and precursors
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b-lactam resistance in Gram-negative rods
Factors that increase the
MIC (resistance)
– Increased enzymatic
inactivation
• High VMAX and/or low KM
• Increased enzyme
concentration
– Decreased intracellular
concentration
• Decreased influx
• Increased efflux)
– Multiple mechanisms may
function in the same strain
PM
OM
PG
porin

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Gram-negative TEM-1 b-lactamases

20-30% of E. coli are ampicillin-resistant
– Most contain a plasmid-encoded class 2b blactamase (TEM-1).
– Active against penicillins but not 3rd generation
cephalosporins. Inhibited by clavulanate.

All K. pneumoniae are ampicillin resistant
– Contain chromosomal SHV-1 (related to TEM-1)


Most E. coli and K. pneumoniae are
susceptible to 1st gen cephs (e.g. cefazolin).
Until recently, all were susceptible to 3rd gen
cephalosporins (e.g. ceftriaxone, ceftazadime).
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Extended Spectrum Beta-lactamases
(ESBLs)

Changes in 1-5 amino acids near active site
serine of TEM-1 (or SHV-1) greatly increase
activity against 3rd gen cephalosporins and
monobactams.
– TEMs 3-29, SHVs 2-6; still inhibited by clavulanate
– Carbapanems are only reliable b-lactams vs ESBL
producers
– Mainly seen in E. coli and K. pneumoniae
– Located on transferable plasmids that may carry
additional resistance genes
26
Ceftazidime, Imipenem and ESBLs

During early 1990s, ESBL-producing Klebsiella became
increasingly common at a hospital in NYC. In 1996
cephalosporin use was sharply curtailed to attempt
reduce the ESBL burden (JAMA 1998;280:1233-37)
1995
1996
Ceftazidime
383
66
Imipenem
197
474
Ceftazidime-resistant Klebsiella
(nososcomial)
150
84
Imipenem-resistant P. aeruginosa
(nosocomial)
67
113
Median monthly
use (grams)
27
ampC b-lactamases

Several Enterobacteriaceae, including
Enterobacter, Citrobacter , and Serratia,
contain an inducible, chromosomal gene
coding for a b-lactamase (ampC)
– Very active in vitro against 1st gen cephs; low
activity against 3rd gen cephs; not inhibited by
clavulanate
– These organisms are naturally resistant to
cefazolin, cefoxitin (strong inducers of ampC)
– Usually sensitive to 3rd gen cephs (poor inducers
of ampC)
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Regulation of ampC


Recycling of peptidoglycan produces NAM-tripeptide
Normally catabolized by AmpD (NAM-tripeptide amidase) and
recycled into new peptidoglycan
Peptidoglycan
Peptidoglycan
autolysins
AmpD
+
[AmpR]

[AmpR]+
NAM-tripeptide is also a positive
activator of AmpR
Increases transcription of ampC
+
ampC
b-lactamase
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Resistance due to derepression of ampC

Many strains of Enterobacter and Citrobacter
develop resistance to 3rd gen cephs during
therapy. Resistant variants contain mutations
that inactivate AmpD
– NAM-tripeptide accumulates, causes stable
derepression of ampC
– Increased levels of AmpC b-lactamase inactivates
3rd gen cephalosporins

Resistant strains remain susceptible to
imipenem (a carbapenem)
– Poorly hydrolyzed, targets low copy PBP
30
b-lactam Resistance in P. aeruginosa

Naturally resistant to many antibiotics
– Outer membrane lacks high permeability porins
present in Enterobacteriaceae.
– Pump mechanism actively exports antibiotics

Acquired resistance is common
– Inducible ampC b-lactamase

Imipenem resistance due to mutations that
inactivate porin D2 (basic AA transporter)
– Sole transporter of imipenem
– Mutations in D2 decrease imipenem influx; blactamase inactivates sufficient drug to confer
resistance.
31
Quinolones

Inhibit topoisomerases/DNA synthesis
– Trap enzyme-DNA complex after strand breakage
– DNA gyrase (topo II) (gyrA/gyrB)
• Primary target in Gram-negatives
– Topoisomerase IV [parC/parE (grlA/grlB in S.aur)]
• Primary target in Gram-positives

Acquired resistance
– Mutations in DNA gyrase and topo IV subunits
• Mainly gyrA and parC (grlA)
• Stepwise increase in resistance results from sequential
mutations in primary and secondary targets
– Efflux pumps
• P. aeruginosa, S. aureus, S. pneumoniae
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Rapid Appearance of Ciprofloxacin
Resistance in S. aureus

After the introduction of ciprofloxacin in the
late ’80s there was rapid increase in
resistance among MRSA
– Prior to introduction of cipro at the Atlanta VAMC,
0% of MRSA were cipro-resistant. One year after
introduction, 79% of MRSA were cipro-resistant
(JID 1991;163:1279-85).
• More than one clone developed resistance
• One-half of pts had been previously treated with
cipro (given for other infections)
• One year later 91% were resistant
• 13% of MSSA also became cipro-resistant
33
Quinolone-Resistant Campylobacter
jejuni in Minnesota


During 1992-8 resistant isolates increased from 1.3 to
10.2% (NEJM 1999;340:1525-32).
Foreign travel was the major risk factor
– Mexico, Caribbean, Asia
– Prior antibiotic therapy accounted for 15% of resistance

There was also an increase in domestically acquired
resistant isolates that was temporally related to the
introduction of quinolones for treatment of poultry in
1995
– Quinolone-resistant isolates were cultured from 20% (18/91)
of retail chicken products
– 6/7 resistant subtypes (PCR-RFLP) from chicken were also
isolated from humans
34
Optimism and Concern on Many Fronts


NEJM (8/14/97): “In Finland, after nationwide
reductions in the use of macrolide antibiotics for
outpatient therapy, there was a significant decline in
the frequency of erythromycin resistance among group
A streptococci isolated from throat swabs and pus
samples.”
NEJM (9/4/97): “We report high-level resistance to
multiple antibiotics, including all the drugs
recommended for plague prophylaxis and therapy, in a
clinical isolate of Y. pestis. The resistance genes were
carried by a plasmid that could conjugate to other Y.
pestis isolates.”
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