Mechanisms of Antimicrobial Resistance Jing-Jou Yan, M.D. Department of Pathology National Cheng Kung University Hospital 26/12/2007

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 :20-51; doi: /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 cells Colonization and persistent infection e.g. bile-resistant in Salmonella and E. coli

Altering or protecting drug targets

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 targets Modification or degradation of drugs Alternative metabolic pathways to bypass the antimicrobial action

Mechanisms of Resistance to β-Lactams

β-Lactam antimicrobials Penicillins Cephalosporins Monobactams Carbapenems

β-Lactam antimicrobials Penicillins  Natural: benzylpenicillin, phenoxymethyl penicillin  Semisynthetic Penicillinase resistant Extended spectrum Aminopenicillins: ampicillin, amoxicillin Carboxypenicillin: carbenicillin, ticarcillin Ureidopenicillins: azlocillin, mezlocillin, piperacillin Cephalosporins Monobactams Carbapenems

β-Lactam antimicrobials Penicillins Cephalosporins  Narrow spectrum (first generation): cephalothin  Expanded spectrum (second generation): cefuroxime, cefoxitin, cefmetazole  Broad spectrum (third generation): cefixime, cefotaxime, ceftazidime, ceftriaxone  Extended spectrum (fourth generation): cefepime, cefpirome Monobactams Carbapenems

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 membrane(s)  Active efflux

Permeability changes Role of outer membranes in β-lactam resistance in E. coli MIC (mg/L) of: E. coliCefoxitinAmpicillinCefazolin Control222 OmpC (-)222 OmpF (-)882 OmpC(-), OmpF (-) Jaffe et al Antimicrob Agents Chemother

Active efflux pumps MIC (mg/L) CiprofloxacinCarbenicillin ΔmexAB-OprM 64  0.03  MexAB-OprM64  256 Role of efflux pump-mediated resistance in P. aeruginosa

Mechanisms of Resistance to β-Lactams Decreased drug accumulation  Permeability changes: loss of outer membrane(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

No. of isolates % of isolates Hsueh PR et al. Emerg Infect Dis 2002 Trends of Penicillin Nonsusceptibility S. pneumoniae, Disk Method, NTUH,

PBP alterations in pneumococci PBPs in pneumonocci: PBP1a/1b, PBP2a/2b/2x, PBP3 Low-level resistance  Mosaic gene formation of each of PBP1a and PBP2a/2b/2x Right-level resistance  Mosaic gene formation of three of PBP1a and PBP2a/2b/2x

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

Hydrolysis of β-lactams by β-lactamases + + β-lactam degraded β-lactamPBP β-lactamase

 -Lactamases Conferring Resistance to Extended-Spectrum  - Lactams in Gram-Negative Bacilli in Taiwan ESBL, extented-spectrum β-lactamase; MBL, metallo- β-lactamase

% Trend in  -lactamases involved in resistance to extended-spectrum  -lactams in K. pneumoniae at NCKUH CMY-2

Trend in  -lactamases involved in resistance to extended-spectrum  -lactams in E. coli at NCKUH

ESBLs in Proteus mirabilis in NCKUH Wu JJ et al. Diagn Microbiol Infect Dis (in press)

Species and the presence of ESBL or AmpC a No. (%) of isolates with ESBLs or AmpC enzymes from hospital: Total N1N2C1C2C3SE E. coli CMY-2-like57 (73.1) 0 (0)40 (47.6)13 (31.7) 8 (44.4) 1 (5.3) 8 (44.4)127 (43.6) CTX-M21 (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 group16 (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-like10 (12.8) 3 (9.1) 5 (6.0) 4 (9.8) 2 (22.2) 3 (15.8) 0 (0) 27 (9.3) None b 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 CMY-2-like 4 (6.9) 1 (1.7) 4 (5.1) 0 (0) 1 (2.7) 0 (0) 10 (3.5) DHA-1-like17 (29.3) 1 (1.7) 5 (6.4) 1 (5.6) 3 (13.0) 4 (10.8) 0 (0) 31 (11.0) CTX-M21 (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 group12 (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) SHV34 (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-like34 (58.6)34 (57.6)17 (21.8) 0 (0) 7 (30.4)30 (81.1) 4 (44.4)126 (44.7) None b 2 (3.4) 1 (1.7) 1 (1.3) 1 (5.6) 0 (0) 5 (1.8) Distribution of ESBLs and AmpC in E. coli and K. pneumoniae in Taiwan Yan et al 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

Methicillin-resistant S. aureus at NCKUH, 1990 and 1998 % Huang et al J Hosp Infect

Methicillin (Oxacillin) Resistance in Staphylococcus aureus Causing Nosocomial Infections NTUH, No. of strains % 2001 Hsueh PR et al. Emerg Infect Dis 2002

Emergence and Spread of Antimicrobial Resistance Genetic alteration Genetic exchange Selective pressure

Emergence and Spread of Antimicrobial Resistance Genetic alterations  Evolution from existing biosynthetic enzymes  Increased spectrum of substrates Genetic exchange Selective pressure

Evolution from Existing Biosynthetic Enzymes PBPs  β-lactamases Aminoglycoside 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 alterations Genetic exchange: transformation, transduction, conjugation  Plasmids  Bacteriophages  Insertion sequences  Transposons  Integrons …. Selective pressure

Integron PPPvB S BH HC ScKH E 0.5 kb intI1 Δ 1 IRibla IMP-8 aac(6’)-Ib catB4qacEΔ1/sul1 Gene cassettes 5‘-CS 3‘-CS

Types of Acquired AmpC CMY-1-related & MOX: close to Aeromonas AmpC CMY-2-related & LAT: close to Citrobacter freundii 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 bla CMY-2 among Salmonella, E. coli, and K. pneumoniae Yan JJ et al. EID 2003 S E K

Emergence and Spread of Antimicrobial Resistance Genetic alterations Genetic exchange Selective pressure

Extent of Antibiotic Use Taiwan, 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: Hsueh PR, NTUH

Emergence of fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis Chiu CH et al. N Engl J Med 2002;346:413-9.

Food animals Meat products Hospitalized patients Humans in community Hospital admission Feces CMY-2 –E. coli And K. P. CMY-2 - E. coli ?? CMY-2 - Salmonella CMY-2 - E. coli ??? CMY-2 - E. coli Widespread distribution of CMY-2-producing E. coli in and outside healthcare settings in Taiwan

Trend in Erythromycin-Resistant group A streptococci Yan et al 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: Please your proposal to me by Jan. 7, 2008.