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 (jingjou@mail.ncku.edu.tw) by Jan. 7, 2008.