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					Mechanisms of
Antibiotic Resistance

Herman Goossens
University of Antwerp, Belgium
University of Leiden, The Netherlands
    Antibiotic Resistance – A Global Problem


                 PRP             VRE                 VISA

     MRSA                ESBL             MBL                   VRSA




     1961       1967     1983    1986    1988        1996       2002

                                             Vancomycin
        Penicillin       Vancomycin              and
                                             teicoplanin
All                3rd gen                                 Vancomycin
                                Carbapenem                     and
-lactams       cephalosporin
                                                           teicoplanin

                     Emergence → Spread
2
    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 genes
       – 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
3
    Resistance by Spontaneous
    Mutations




4
    Acquisition of Resistance Genes




5
    Mechanisms of Resistance Gene Transfer




6
    Mechanisms of Resistance Gene
    Transfer: Transposons




7
    Mechanisms of Resistance Gene
    Transfer: Integrons




8
    Mechanisms of Resistance

    • Antibiotics exert selective pressure that favours
      emergence of resistant organisms
    • Bacteria employ several biochemical strategies to
      become resistant




9
     Major Classes of Antibiotics
                                                            Major resistance
  Antibiotic                    Mechanism of action         mechanisms
  β-Lactams                     Inactivate PBPs             • β-lactamases
                                (peptidoglycan synthesis)   • Low affinity PBPs
                                                            • Efflux pumps
  Glycopeptides                 Bind to precursor of        • Modification of precursor
                                peptidoglycan
  Aminoglycosides               Inhibit protein synthesis   • Modifying enzymes (add
                                (bind to 30S subunit)         adenyl or Phosphate)
  Macrolides                    Inhibit protein synthesis   • Methylation of rRNA
                                (bind to 50S subunit)       • Efflux pumps
  (Fluoro)Quinolones            Inhibit topoisomerases      • Altered target enzyme
                                (DNA synthesis)             • Efflux pumps

10 PBPs penicillin-binding proteins
     -Lactams: Classification (1)

     • Penicillins
        – Narrow-spectrum penicillins
        – Broad-spectrum penicillins
        – β-lactamase inhibitor combinations
        – Oxacillin derivatives
     • Cephalosporins (ATC/WHO 2005 classification)
        – 1st generation: Gram-positive cocci (GPCs), some Gram-
          negative bacilli (GNBs)
        – 2nd generation: some GNBs, anaerobes
        – 3rd generation: many GNBs, GPCs
11      – 4th generation: many GNBs resistant to 3rd generation, GPCs
     -Lactams: Classification (2)

     • Carbapenems
       – Imipenem, meropenem, ertapenem
     • Monobactams
       – Aztreonam




12
     Mechanism of Action of -Lactams (1)

     • Structure of peptidoglycan
                                Cytoplasm



     NAG-NAM-NAG-NAM
           |
         L-Ala       NAG-NAM-NAG-NAM
           |                |
         D-               L-Ala
         Glu                |
           |   -(AA)n-NH2 D-Glu
         L-diA              |
           |              L-diA-(AA)n-
         D-Ala              | NH2
           |              D-Ala
         D-Ala              |
                          D-Ala
 Transpeptidation
 reaction
13
     Mechanism of Action of -Lactams (2)

     • Penicillin-binding proteins (PBPs)
        – Membrane-bound enzymes
        – Catalyse final steps of peptidoglycan synthesis
          (transglycosylation and transpeptidation)
     • -lactams
        – Act on PBPs, inhibit transpeptidation
        – Substrate analogues of D-Ala-D-Ala




14
     Resistance to -Lactams




15
     Resistance to -Lactams

     • Gram-negative -lactamases
        – Major resistance mechanism in nosocomial GNB pathogens
        – >470 -lactamases known to date
        – Classified into 4 groups based on sequence similarity
             • Ambler Class A (TEM, SHV, CTX), C and D (OXA) are
               serine -lactamases
             • Ambler Class B are metallo--lactamases
        – Their spread has been greatly exacerbated by their integration within
          mobile genetic elements
        – Integron-borne -lactamase genes are part of multi drug resistance
          gene cassettes


 Multidrug-resistant nosocomial pathogens Selection of potent -lactamases
16
 with complex resistance patterns         through use of non--lactam agents
     Ambler Classification of β-Lactamases


                               β-lactamases



Active site     Serine-enzymes             Zinc-enzymes


Nucleotide
sequence           A     C      D                  B

              Four evolutionarily distinct molecular classes
17
   Modified Bush–Jacoby–Medeiros
   Classification of –Lactamases

   Functional Substrate profile   Molecular   Inhibitor    Example
   Group                          Class


   1      Cephalosporinase        C           Oxa         AmpC, MIR-1

   2a     Penicillinase           A           Clav.       S.aureus
   2b     Broad spectrum          A           Clav.       TEM-1/2, SHV-1
   2be    Extended spectrum       A           Clav.       TEM 3-29, TEM46-104 SHV2-
                                                          28, CTX-M types
   2br    Inhibition resistant    A           -           TEM 30-41 (IRT1-12)
   2c     Carbenicillinase        A           Clav.       PSE-1

   2d     Oxacillinase            D           (Clav.)     OXA-1 (OXA-2 &-10 derived
                                                          ESBL)
   2e     Cephalosporinase        A           Clav.       FPM-1 P. vulgaris, CepA B.
                                                          fragilis.
   2f     Carbapenemase           A           Clav.       IMI-1, NmcA, Sme 1-3
   3      Metallo-enzyme          B           -           S.maltophilia
18 4      Penicillinase           -           -           B.cepacia
      -Lactamases: Classification

          Serine enzymes                    Metallo (Zn) enzymes



Group C      Group A         Group D                                  Group B

AmpC        TEM/SHV            OXA                                   IMP/VIM

 Cephs     Pens, Cephs     Pens, esp Oxa                   Carbapenems
Inhib-R      Inhib-S         Inhib-R/S                           Inhib-R

 19                            Bush. Rev Inf Dis 1987;10:681; Bush et al. Antimicrob Agents Chemother
                                               1995;39:12; Bush. Curr Opin Investig Drugs 2002;3:1284
       Induction of Group 1 (AmpC)
       -Lactamases


                                                        Inducible:
                                                        Enterobacter spp.
                                                        Citrobacter spp.
Amount                                                  Morganella spp.
enzyme                                                  Providencia spp.
                                                        Serratia spp.
per cell                                                P. aeruginosa


                                               Absent :
                                               Salmonella spp.
                               Basal :
                                               Klebsiella spp.
                               E. coli
                               Shigella spp.




                 -lactam concentration
  20
     Selection of Group 1 (AmpC)
     -Lactamases
                     Population of inducible organisms




                     Derepressed cell due to ampD mutation
                     (Enterobacter : 1 of 105 !)

                     Ceftazidime, ceftriaxone, piperacillin, etc.:

                     Selection of derepressed cell




                     Multiplication and spread of derepressed
                     clone
21
     Group 1 (AmpC) -Lactamases

     • Produced constitutively in tiny concentrations by certain GNB
     • Induction of production:
         – Can occur by the exposure to certain antibiotics (eg, carbapenems)
         – Only in vitro phenomenon; not clinically relevant (stops when antibiotic
           use is discontinued; carbapenems not affected by these enzymes)
     • Selection of production:
         – Can occur by the use of certain antibiotics (eg, ceftazidime)
         – Also in vivo phenomenon; highly clinically relevant (does not stop when
           antibiotic use is discontinued; leads to selection and spread of ABR
           clones)
     • Therapeutic options:
         – 4th generation cephalosporins (but resistance may occur with minor AA
           changes)
         – Carbapenems
22
     Resistance to -Lactams

     • Chromosomal AmpC -lactamases
        – Several Enterobacteriaceae, including Enterobacter, Citrobacter, and
          Serratia contain an inducible, chromosomal gene coding for a -
          lactamase
        – Resistant to cephalosporins and monobactams; not inhibited by
          clavulanate; Class C -lactamases
     • Plasmid-mediated AmpC -lactamases
        – Arose through transfer of AmpC chromosomal genes into plasmids
        – Not inducible, with substrate profile (usually) same as parental enzyme
        – Highly prevalent in the naturally AmpC-deficient K. pneumoniae
        – Emergence predominantly in community-acquired infections
          (Salmonella spp., E. coli)
        – Co-resistance to aminoglycosides, SXT, quinolones
        – Wide dissemination worldwide (SE Asia, N Africa, South Europe, USA)
23
     Plasmid-mediated AmpC -lactamases (1)
     Enzyme   Host                    Country   Year isolated
     MIR-1    K. pneumoniae           US        1988
     ACT-1    K. pneumoniae           US        1994
              E. coli
     BIL-1    E. coli                 UK        1989
     CMY-2    K. pneumoniae           Greece    1990
              S. senftenberg          France    1994
              Salmonella              US        1996
              E. coli                 Libya     1996
              Salmonella              Spain     1999
              Salmonella              Romania   2000
     LAT-1    K. pneumoniae           Greece    1993
     LAT-2    K. pneumoniae,          Greece    1994
              E. coli, E. aerogenes
     CMY-3    P. mirabilis            France    1998
     CMY-4    P. mirabilis            Tunisia   1996
              E. coli                 UK        1999
              K. pneumoniae           Sweden    1998
     CMY-5    K. oxytoca              Sweden    1988
24   CMY-7    E. coli                 India
     Plasmid-mediated AmpC -lactamases (2)

      Enzyme   Host                Country        Year isolated
      DHA-1    Salm. enteritidis   Saudi Arabia   1992
               K. pneumoniae       Taiwan         1999
                                   US             1996-2000
      DHA-2    K. pneumoniae       France         1992
      ACC-1    K. pneumoniae       Germany        1997
               K. pneumoniae       France         1998
               P. mirabilis        Tunisia        1997
               K. pneumoniae       Tunisia        1999
               Salm. livingstone   Tunisia        2000




25
     Plasmid-mediated AmpC -lactamases (3)

      Enzyme   Host            Country     Year isolated
      FOX-1    K. pneumoniae   Argentina   1989
      FOX-2    E. coli         Germany     1993
      FOX-3    K. oxytoca,     Italy       1994
               K. pneumoniae
      FOX-4    E. coli         Canaries    1998
      FOX-5    K. pneumoniae   US          1999
      CMY-1    K. pneumoniae   Korea       1989
      CMY-8    K. pneumoniae   Taiwan      1998
      CMY-9    E. coli         Japan       1995
      CMY-10   E. aerogenes    Korea       1999
      CMY-11   E. coli         Korea       1998
      MOX-1    K. pneumoniae   Japan       1991
      MOX-2    K. pneumoniae   France      1999



26
     Resistance to -Lactams

     • Extended-spectrum -lactamases (ESBL)
        – No consensus of the precise definition of ESBLs
        – In general: β-lactamases conferring resistance to the
          penicillins, 1st , 2nd, 3rd, and even 4th generation
          cephalosporins, and monobactams, not to carbapenems
          and cephamycins
        – Inhibited by -lactamase inhibitor clavulanic acid
        – Derived from Class A -lactamases (exceptions are Class
          D, OXA): TEM, SHV, CTX-M, OXA, VEB, PER,...
        – Differ from their progenitors by 1–5 amino acids
        – Marked and unexplained predilection for Klebsiella
          pneumoniae
        – Therapeutic options: carbapenems
27
     The Story of E. coli Resistant to Ampicillin

     • June 1964: ampicillin released in Europe
     • December 1964; the first case of ampicillin- resistant
       E. coli detected
     • Mrs Temoneira (Athens, Greece):
        – Urinary isolate of E. coli
        – Produced -lactamase (TEM-1)
        – Genes encoding the -lactamase found on a plasmid




28
     The Story of Klebsiella Universally
     Resistant to Ampicillin

     • SHV-1 enzyme: -lactamase with a narrow spectrum
       of activity (ampicillin)
     • Chromosomally encoded
     • If produced in high amounts:
        – May result in resistance to cefazolin and piperacillin
        – May even overcome β-lactamase inhibitors (clavulanic acid
          or tazobactam)




29
     The Story of ESBL-producing
     Enteric GNB
     • Third generation cephalosporins:
        – Developed in response to proliferation of K. pneumoniae
          and E. coli producing -lactamases active against ampicillin
          and first generation cephalosporins
        – Introduced in Europe in the early 1980s
     • Emergence of extended-spectrum -lactamases:
        – Cefotaxime marketed in Germany in September 1981
        – Cefotaxime-resistant Klebsiella isolate detected in Frankfurt
          in March 1982 (mutant of the gene encoding SHV-1)


30
     Evolution of TEM Enzymes
                                      MIC (mg/mL) ceftazidime
                 102         162
TEM-1                                          0.25


               glutamine   arginine


TEM-12
                                                2.0
               glutamine   serine



TEM-26
                                                128
                lysine     serine
31
     ESBLs in Non-fermenters

     • Emergence of transferable ESBL enzymes (Class A, B or D) in
       non-fermenters (P. aeruginosa, Acinetobacter spp.)
     • ESBL types often different (PER-1, VEB-1, OXA,…) from
       Enterobacteriaceae
     • Multiple resistance mechanisms co-expressed (chromosomal
       AmpC -lactamase, impermeability, efflux)
     • Non-fermenters should not be tested routinely for ESBLs
        – P. aeruginosa: «False-negative» (most ESBLs not inhibited by
          clavulanate)
        – Acinetobacter spp.: «False-positive» DD with clavulanate (intrinsic
          activity of -lactam inhibitors)
        – S. maltophilia: «False-positive» DD with clavulanate (inhibition of L2
          chromosomal enzyme)
32
     FREQUENTLY MADE COMMENT:

     ―I don’t see ESBLs in my hospital‖




33
     Current ESBL Detection Methods Fail

     • Routine tests are not designed for ESBL detection
     • Low level ESBL expression will not be detected by
       current tests using low inoculum
     • MIC values and zone sizes of ESBL producers overlap
       those of susceptible non-ESBL producers
     • ESBL double disk test may be inaccurate if positioning
       is suboptimal
     • ESBL breakpoint methods are limited since MICs for
       different strains can range over 7 dilutions

34
     Resistance to -Lactams

     • Carbapenemases
       – Defined as -lactamases, hydrolyzing at least imipenem
         or/and meropenem or/and ertapenem
       – Belong to Ambler Class A, B, and D, of which Class B are
         the most clinically significant:
          • Class A: KPC, SME & NMC/IMI
          • Class B: IMP, VIM & SPM metallo -lactamases
          • Class D: OXA-23, -40 & -58 related




35
     Class B (Metallo)-Carbapenemases

     • Hydrolyzing virtually all -lactams
     • Mediate broad spectrum -lactam resistance
     • No clinical inhibitor available
     • Present on large plasmids and integrons
     • Genes are continuously spreading
     • Associated (80%) with aminoglycoside resistance


       Still rare but increasing, especially in non-fermenters

36
     Mobile Carbapenamases
                              Class I integron

                                     blaIMP
            ORF1      aacC4          blaVIM            aacC1
 5'cs                                                               3'cs


     • Nosocomial outbreak of carbapenem-resistant P.aeruginosa
       and A. baumanii reported in Canada and France, respectively
     • Cross-resistance to other beta-lactams and to other AB classes
     • Link with aminoglycoside use, not necessarily carbapenems!


37
     Mobile Class B -Lactamases
Enzyme Host              Country (Year)    Enzyme Host                Country (Year)
IMP-1  S. marcescens     Japan (>91)       VIM-1  P.aeruginosa        Italy    (1997)
       P. aeruginosa     Japan                    A. baumannii        Italy    (1997)
       A. xylosoxydans   Japan                    P.aeruginosa        Greece (1996)
       P. putida         Japan                    E. coli             Greece (2001)
       C. freundii       Japan                    A. xylosoxydans     Italy    (1997)
       K. pneumoniae     Japan,            VIM-2  P. aeruginosa       France (1996)
                         Singapore (99)           P. aeruginosa       Greece (1996)
         A. baumannii    Japan                    P. aeruginosa       Italy    (1998)
         P. stutzeri,    Taiwan                   S. marcescens       Korea (2000)
         P. putida                                A. baumannii        Korea (1998)
         A. junii        UK       (00)            P. aeruginosa       Belgium (2004/5)
IMP-2    A. baumannii    Italy    (97)            P.putida stutzeri   Taiwan (>1997)
IMP-3    S. flexneri     Japan (96)        VIM-3  P. aeruginosa       Taiwan (>1997)
IMP-4    Acinetobacter   Hong Kong (>94)   VIM-4  P. aeruginosa       Greece (2001)
         C. youngae      China    (98)
IMP-5    A. baumannii    Portugal (98)
IMP-6    S. marcescens   Japan (96)        SPM-1   P. aeruginosa      Brazil   (1997)
IMP-7    P. aeruginosa   Canada (95)
                         Malaysia (99)
                                           GIM-1   P. aeruginosa   Germany (2003)
IMP-8    K. pneumoniae Taiwan (98)
IMP-9    P. aeruginosa   China    (?)
IMP-10   A. xylosoxydans Japan (00)
         P. aeruginosa   Japan (97)
38
     Class D Oxacillinase — Carbapenemases

     • Class D enzymes
     • OXA-23, -24, -25, -26, -27, -28, -40, -49, -58, ….
     • Highly mobile (integron, plasmid)
     • Found in South America, South-East Asia, Europe
       (Greece, Spain, Portugal, France, Belgium)
     • Multi-drug resistance (penicillins and 3rd & 4th
       generation cephalosporins, BL/BL-inhibitors,
       aminoglycosides, SXT,…)
     • Variable resistance levels to imipenem and
       meropenem (4–>256 mg/mL)
39
        Rapidly Increasing Antibiotic
      Resistance Constitutes One of the
           Most Important Clinical,
     Epidemiological and Microbiological
             Problems of Today


40

				
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