Mechanisms of Antimicrobial Resistance Jing-Jou Yan, M.D. Department of Pathology National Cheng...
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Transcript of Mechanisms of Antimicrobial Resistance Jing-Jou Yan, M.D. Department of Pathology National Cheng...
Mechanisms of Antimicrobial Resistance
Jing-Jou Yan, M.D.
Department of PathologyNational Cheng Kung University Hospital
26/12/2007
Action of antimicrobials
Action of antimicrobials Inhibition of cell wall synthesis
β-lactams, vancomycin Inhibition of DNA synthesis
Quinolones Inhibition of protein synthesis
50S inhibitors: erythromycin 30S inhibitors: aminoglycosides
Overview of Mechanisms of Antimicrobial Resistance
Decreased drug accumulation by permeability changes
Decreased drug accumulation by active efflux
A major contribution to intrinsic antibiotic resistance in Gram-negative species: broad-specificity drug-efflux pumps.
Copyright restrictions may apply.
Poole, K. J. Antimicrob. Chemother. 2005 56:20-51; doi:10.1093/jac/dki171
Schematic diagram of representative drug exporting systems in Gram-negative bacteria, highlighting the different families of pumps involved in resistance
ATP-binding cassette (ABC) superfamily
Major facilitator (MF) superfamily
Multidrug and toxic-compound extrusion (MATE) family
Small multidrug resistance (SMR) family
Resistance nodulation division (RND) family
Efflux pumps and pathogenicity
Adherence to and invasion of host cellsColonization and persistent infection
e.g. bile-resistant in Salmonella and E. coli
Altering or protecting drug targets
Modification or degradation of drugs
Modification or degradation of drugs
Modification or degradation of drugs
Alternative metabolic pathways to bypass the antimicrobial action
Overview of Mechanisms of Antimicrobial Resistance
Decreased drug accumulation Permeability changes Active efflux
Altering or protecting drug targetsModification or degradation of drugsAlternative metabolic pathways to
bypass the antimicrobial action
Mechanisms of Resistance to β-Lactams
β-Lactam antimicrobials
PenicillinsCephalosporinsMonobactamsCarbapenems
β-Lactam antimicrobials
Penicillins Natural: benzylpenicillin, phenoxymethyl penicillin Semisynthetic
Penicillinase resistant Extended spectrum
• Aminopenicillins: ampicillin, amoxicillin
• Carboxypenicillin: carbenicillin, ticarcillin
• Ureidopenicillins: azlocillin, mezlocillin, piperacillin
CephalosporinsMonobactamsCarbapenems
β-Lactam antimicrobials
PenicillinsCephalosporins
Narrow spectrum (first generation): cephalothin Expanded spectrum (second generation): cefuroxime, cefoxi
tin, cefmetazole Broad spectrum (third generation): cefixime, cefotaxime, ceft
azidime, ceftriaxone Extended spectrum (fourth generation): cefepime, cefpirome
MonobactamsCarbapenems
Action of β-Lactams
Targets: D-alanyl-D-alanine trans- and carboxypeptidases (PBPs) sugar chains cross-linked by peptides
Action: PBPs form acyl esters with β-lactams
Mechanisms of Resistance to β-Lactams
Decreased drug accumulation Permeability changes: loss of outer membran
e(s) Active efflux
Permeability changes
Role of outer membranes in β-lactam resistance in E. coli
MIC (mg/L) of:
E. coli Cefoxitin Ampicillin Cefazolin
Control 2 2 2
OmpC (-) 2 2 2
OmpF (-) 8 8 2
OmpC(-), OmpF (-)
128 16 64
Jaffe et al. 1982 Antimicrob Agents Chemother
Active efflux pumps
MIC (mg/L)
Ciprofloxacin Carbenicillin
ΔmexAB-OprM 64 0.03 64 0.03
MexAB-OprM 64 256 64 256
Role of efflux pump-mediated resistance in P. aeruginosa
Mechanisms of Resistance to β-Lactams
Decreased drug accumulation Permeability changes: loss of outer membran
e(s) Active efflux
Altering or protecting drug targets: PBP alterations
Modification of normal PBPs by mosaic gene formation
Susceptible PBP Resistant PBP
Mosaic geneSusceptible gene
0
10
20
30
40
50
60
70
80
90
100
0
50
100
150
200
250
300
350
No. of isolates
PNSSP
No
. o
f is
ola
tes
No
. o
f is
ola
tes
% o
f is
ola
tes
Hsueh PR et al. Emerg Infect Dis 2002
Trends of Penicillin NonsusceptibilityS. pneumoniae, Disk Method, NTUH, 1984-2001
PBP alterations in pneumococci
PBPs in pneumonocci: PBP1a/1b, PBP2a/2b/2x, PBP3
Low-level resistance Mosaic gene formation of each of PBP1a and P
BP2a/2b/2x
Right-level resistance Mosaic gene formation of three of PBP1a and P
BP2a/2b/2x
Mechanisms of Resistance to β-Lactams
Decreased drug accumulation Permeability changes: loss of outer membran
e(s) Active efflux
Altering or protecting drug targets: PBP alteration
Modification or degradation of drugs: production of β-lactamases
Hydrolysis of β-lactams by β-lactamases
+
+
β-lactam
degraded β-lactam PBP β-lactamase
Bush-Jacoby-Medeiros functional classification of β -Lactamases
Group
Molecular
class
Characteristics
1 C Cephalosporinase not inhibited by clavulanic acid 2a A Penicillinases inhibited by clavulanic acid
2b A Broad-spectrum enzymes inhibited by clavulanic acid 2be A Extended-spectrum enzymes inhibited by clavulanic acid
(ESBLs) 2br A Broad-spectrum enzymes with reduced binding to clavulanic
acid (IRTs) 2c A Carbenicillin-hydrolyzing enzymes inhibited by clavulanic acid 2d D Cloxacillin-hydrolyzing enzymes inhibited by clavulanic acid 2e A Cephalosporinases inhibited by clavulanic acid 2f A Carbapenem-hydrolyzing nonmetallo--lactamases 3 B Metallo--lactamases (MBLs)
4 NDb Penicillinases not inhibited by clavulanic acid
-Lactamases Conferring Resistance to Extended-Spectrum -Lactams in Gram-Negative Bacilli in Taiwan
-lactamase Species Location ESBL SHV Enterobacteriaceae Plasmid CTX-M Enterobacteriaceae Plasmid AmpC CMY-2 E. coli, K. pneumoniae,
Salmonella spp. Plasmid
CMY-8 K. pneumoniae Plasmid DHA-1 K. pneumoniae, E. coli Plasmid MBL IMP-1 P. putida, P. stutzeri Chromosome IMP-8 K. pneumoniae, K. oxytoca,
E. cloacae Plasmid
VIM-2 P. putida, P. stutzeri, C. freundii
Chromosome, plasmid
VIM-3 P. aeruginosa Chromosome
ESBL, extented-spectrum β-lactamase; MBL, metallo- β-lactamase
01
2345
678
9101112
1314
1999 2000 2001 2002
SHV CTX-M CMY-1 CMY-8 DHA-1 IMP-8 ESBL AmpC%
Trend in -lactamases involved in resistance to extended-spectrum -lactams in K. pneumoniae at NCKUH
CMY-2
0
1
2
3
4
5
6
1999 2000 2001 2002
SHV CTX-M TEM ESBL CMY-2
Trend in -lactamases involved in resistance to extended-spectrum -lactams in E. coli at NCKUH
0.7% 0.7%
3.7%
1.7%
2.6%
0.8%
0.7%
1.2%
0.6%3.9%
3.6%
1.2%0.5%
0.6%
0.0%
1.0%
2.0%
3.0%
4.0%
5.0%
6.0%
7.0%
1999 2000 2001 2002 2003 2004 2005
Year
% o
f C
TX
-M p
rod
uce
rs
% of CTX-M-66 producers
% of CTX-M-24 producers
% of CTX-M-14 producers
% of CTX-M-3 producers
ESBLs in Proteus mirabilis in NCKUH 1999 - 2005
Wu JJ et al. Diagn Microbiol Infect Dis (in press)
Species and the presence of ESBL or AmpCa
No. (%) of isolates with ESBLs or AmpC enzymes from hospital:
TotalN1 N2 C1 C2 C3 S E
E. coli 78 33 84 41 18 19 18 291
CMY-2-like 57 (73.1) 0 (0) 40 (47.6) 13 (31.7) 8 (44.4) 1 (5.3) 8 (44.4) 127 (43.6)
CTX-M 21 (26.9) 28 (84.8) 47 (56.0) 28 (68.3) 8 (44.4) 17 (89.5) 9 (50.5) 158 (54.3)
CTX-M-1 group 5 (6.4) 4 (12.1) 19 (22.6) 5 (12.2) 2 (22.2) 2 (10.5) 4 (22.2) 41 (14.1)
CTX-M-9 group 16 (20.5) 24 (72.7) 28 (33.3) 23 (56.1) 6 (33.3) 15 (78.9) 5 (27.8) 117 (40.2)
SHV-5-like 10 (12.8) 3 (9.1) 5 (6.0) 4 (9.8) 2 (22.2) 3 (15.8) 0 (0) 27 (9.3)
Noneb 1 (1.3) 3 (9.1) 4 (4.8) 1 (2.4) 3 (16.7) 0 (0) 2 (11.1) 14 (4.8)
K. pneumoniae 58 59 78 18 23 37 9 282
CMY-2-like 4 (6.9) 1 (1.7) 4 (5.1) 0 (0) 0 (0) 1 (2.7) 0 (0) 10 (3.5)
DHA-1-like 17 (29.3) 1 (1.7) 5 (6.4) 1 (5.6) 3 (13.0) 4 (10.8) 0 (0) 31 (11.0)
CTX-M 21 (36.2) 21 (35.6) 67 (85.9) 15 (83.3) 16 (69.6) 10 (27.0) 5 (55.6) 155 (55.0)
CTX-M-1 group 12 (20.7) 7 (11.9) 44 (56.4) 8 (44.4) 14 (60.9) 8 (21.6) 5 (55.6) 98 (34.8)
CTX-M-9 group 9 (15.5) 14 (23.7) 23 (29.5) 7 (38.9) 2 (8.7) 2 (5.4) 0 (0) 57 (20.2)
SHV 34 (58.6) 39 (66.1) 18 (23.1) 1 (5.6) 7 (30.4) 32 (86.5) 4 (44.4) 135 (47.9)
SHV-2-like 0 (0) 5 (8.5) 1 (1.3) 1 (5.6) 0 (0) 2 (5.4) 0 (0) 9 (3.2)
SHV-5-like 34 (58.6) 34 (57.6) 17 (21.8) 0 (0) 7 (30.4) 30 (81.1) 4 (44.4) 126 (44.7)
Noneb 2 (3.4) 1 (1.7) 1 (1.3) 1 (5.6) 0 (0) 0 (0) 0 (0) 5 (1.8)
Distribution of ESBLs and AmpC in E. coli and K. pneumoniae in Taiwan
Yan et al. 2006. Antimicrob Agents Chemother
Mechanisms of Resistance to β-Lactams
Decreased drug accumulation Permeability changes: loss of outer membrane(s) Active efflux
Altering or protecting drug targets: PBP alteration
Modification or degradation of drugs: production of β-lactamases
Alternative metabolic pathways to bypass the antimicrobial action: acquisition of MecA
Bypass resistance: Methicillin-resistant Staphylococcus aureus
PBP2a
Methicillin-resistant S. aureus produces PBP-2a encoded by mecA inserted on chromosome
Methicillin
PBPs
14.117.8
36.8
44.7
49.951.8
54.2
59.9 61.0
0
10
20
30
40
50
60
701989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
Year
% o
f M
RSA
Methicillin-resistant S. aureus at NCKUH, 1990 and 1998
%
Huang et al. 2000 J Hosp Infect
Methicillin (Oxacillin) Resistance in Staphylococcus aureus Causing Nosocomial InfectionsNTUH, 1986-2001
0
100
200
300
400
1986 88 90 92 94 96 98 2000
0
20
40
60
80
100
MRSAMSSA% of MRSA
No
. o
f st
rain
s
%
2001
Hsueh PR et al. Emerg Infect Dis 2002
Emergence and Spread of Antimicrobial Resistance
Genetic alterationGenetic exchangeSelective pressure
Emergence and Spread of Antimicrobial Resistance
Genetic alterations Evolution from existing biosynthetic enzymes Increased spectrum of substrates
Genetic exchangeSelective pressure
Evolution from Existing Biosynthetic Enzymes
PBPs β-lactamasesAminoglycoside modifying enzymes
Protein kinases Aminoglycoside phosphotransferases
Protein acylases aminoglycoside acetyltransferases
Massava & Mobashery 1998 Antimicrob Agents Chemother
Increased spectrum of substrates
Narrow-spectrum β-lactamases extended-spectrum β-lactamases SHV-1 SHV-2 and more TEM-1 & -2 TEM-3 and more
Emergence and Spread of Antimicrobial Resistance
Genetic alterationsGenetic exchange: transformation, transdu
ction, conjugation Plasmids Bacteriophages Insertion sequences Transposons Integrons ….
Selective pressure
IntegronIntegron
P PPv B S Pv B H H C Sc K H H E
0.5 kb
intI1Δ1IRi blaIMP-8 aac(6’)-Ib catB4 qacEΔ1/sul1
Gene cassettes
5‘-CS 3‘-CS
Types of Acquired AmpCTypes of Acquired AmpC CMY-1-related & MOX: close to Aeromonas A
mpC CMY-2-related & LAT: close to Citrobacter fre
undii AmpC FOX: related to Aeromonas AmpC DHA: related to Morganella morganii AmpC ACT: Enterobacter cloacae AmpC ACC: related to Hafnia alvei AmpC
Interspecies spread of blaCMY-2 among Salmonella, E. coli, and K. pneumoniae Yan JJ et al. EID 2003
S E K
Emergence and Spread of Antimicrobial Resistance
Genetic alterationsGenetic exchangeSelective pressure
Extent of Antibiotic UseExtent of Antibiotic Use Taiwan, Before 2001Taiwan, Before 2001
High consumption of antibiotics in the community– 65.4% use in RTI: 1/3 for acute URTI
Inappropriate use of surgical prophylaxis in hospitals (timing and duration)
Extensive use in ICUs
Widespread use in farms and feed mills
Liu YC. Lancet 1999; McDonald LC et al. J Formos Med Assoc 2001; McDonald LC et al. J Microbiol Immunol Infect 2001; Chiu CH et al. N Engl J Med 2002;346:413-9. Hsueh PR, NTUH
Emergence of fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis
Chiu CH et al. N Engl J Med 2002;346:413-9.
Foodanimals
Meatproducts
Hospitalizedpatients
Humans in community
Hospitaladmission
Feces
CMY-2 –E. coliAnd K. P.
CMY-2 - E. coli ??CMY-2 - Salmonella
CMY-2 - E. coli
???
CMY-2 - E. coli
Widespread distribution ofCMY-2-producing E. coli in andoutside healthcare settings in Taiwan
Trend in Erythromycin-Resistant group A streptococci
Yan et al. 2003. J Clin Microbiol
Take-Home Problem
The increasing prevalence of cephalosporin resistance in gram-negative bacilli is causing increased reliance on carbapenems, and the emergence of carbapenem resistance has become a matter of great concert. In National Cheng Kung University Hospital, the first carbapenem-resistant Escherichia coli isolate was noted in 1999, and the prevalence of carbapenem resistance in the bacterial species has increased extremely from then on. Please write a research proposal (limited to one page, English only) to find out genetic alteration(s) that may contribute to carbapenem resistance in such isolates. In the proposal, you should describe at least your hypothesis and strategy(s) of determining the genetic alteration(s).
Ref. Livermore DM, Woodford N. The β-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. TRENDS in Microbiology 2006;14: 413-420
Please e-mail your proposal to me ([email protected]) by Jan. 7, 2008.