Types, mechanism of action, treatment of AmpC

1. Amp C beta lactamases
Dr. Deepa Devhare

2. • Beta lactamases
• Classification of beta lactamases
• Types of Amp C enzymes
• Mechanism of action
• Laboratory diagnosis
• Treatment options

3. Beta lactamases
• Beta-lactamases are a diverse class of enzymes produced by bacteria that
break open the beta-lactam ring, inactivating the beta-lactam antibiotic.

4. Classification of beta lactamases

5. Amp c beta lactamases
• clinically important cephalosporinases in gram negative bacilli
• 1940: first bacterial enzyme reported to destroy penicillin was the
AmpC -lactamase of Escherichia coli
.

7. Types of Amp C beta lactamases
• Inducible: gene needs to be induced/ activated in order to produce
enzyme
• Constitutive: Produces enzyme without induction/ activation of
gene
Chromosomal
• Constitutive or
Inducible
Plasmid encoded
• Mostly
constitutive

8. Chromosomal constitutive Amp c :
 Constitutive low level expression of Amp C
 E.coli
 Shigella
 Enterobacter
 Citrobacter fruendii
 Acinetobacter

9. Chromosomal Inducible Amp c :
• At baseline, no Amp C production
• Amp C production after gene induction by inducing substances like third
generation cephalosporins
 Serratia
 Pseudomonas
 Indole positive proteae
 Citrobacter
 Enterobacter sp

11. Plasmid mediated Amp C beta lactamases (pAmp C)

12. Nomenclature of pAmp C beta lactamases
Amp C gene Name derived from
CMY resistance to cephamycins
FOX resistance to cefoxitin
MOX resistance to moxalactam
LAT resistance to latamoxef
ACT Amp C type
ACC Ambler class C
DHA Daharan hospital Saudi arabia
MIR Miriam Hospital in Providence, Rhode Island.
BIL After the patient name Bilal

13. Plasmid mediated Amp C beta lactamases (pAmp C)
• Mostly constitutive except genes for ACT-1, DHA-1, DHA-2, and CMY-13 are inducible
• Plasmids often carry multiple other resistance genes
 aminoglycosides
 chloramphenicol
 quinolones
 sulfonamide
 tetracycline
 trimethoprim
 TEM-1, CTX-M-3, SHV varieties, and VIM-1
Organisms carrying pAmpC
 Klebsiella pneumoniae
 Salmonella sp
 E.Coli
 Proteus mirabilis

14. Spectrum of action of Amp C
• Resistant to Penicillins
• Resistant to cephamycins
• 1st ,2nd ,3rd generation cephalosporins
• Monobactams
• Not inhibited by beta lactamase inhibitors
Inhibitors of class A enzymes: clavulanic acid, sulbactam, and tazobactam have
much less effect on AmpC -lactamases, although some are inhibited by tazobactam
or sulbactam

16. Extended spectrum Amp C beta lactamases (ESAC)
2007: Nordmann and Mammeri
Structurally related to wild type cephalosporinases
Broader spectrum of resistance esp cefepime and carbapenems
Found in E.coli, Pseudomonas aeruginosa, Acinetobacter

17. What are inducers?
• Antibiotics which induces/starts Amp C enzyme production

18. What are substrates?
• Antibiotics which can be hydrolyzed by AmpC
Good(=Unstable) Variable Poor (=stable )
Penicillins 2nd, 3rd generation
cephalosporins
Imipenem
Cefazolin, cephalothin Aztreonam 4th generation
cephalosporins

19. Inducer-Substrate combinations
Combination Antibiotics Comments
Good substrate + good inducer Amino penicilins, 1st generation
Cephal., Clavulanate
Good inducer + poor substrate Imipenem Remains active as long as other
resistance mechanisms are
absent (porin loss or efflux
pump)
Non inducer+ Good substrate Carboxy ureidopenicillins, 2nd -3rd
generation cephalosporins,
Aztreonam
Remain active against SPICE
organisms in the absence of
other inducing agent
Non inducer+ poor substrate Cefepime
Cefepirome
May get affected in
hyperproduction of Amp C

20. Mechanisms of action of Amp C
Chromosomal
Constitutive low level
Ampc
Amp C de-
repression
Amp C induction
Chromosomal
inducible
Plasmid mediated Amp C Constitutive overproduction

21. Amp G- Absorption of cell wall
fragments
Amp D- Amidase ,removes
degradation products or cell wall
fragments
Amp R – Transcriptional regulator
Cell wall constantly re-cycled

22. Amp C
Induction

23. Amp C
de-repression

24. Amp C in E.coli- Special concern???
• naturally carries a chromosomal ampC (campC) gene- constitutive low-level ampC
expression
• lacks amp R gene, so AmpC production is not inducible
• regulated by promoter and attenuator mechanisms
• allows the use of beta-lactam antibiotics to treat these E. coli infections in the absence of
other resistance mechanisms.
• Over production of Amp C:
1. mutations in the promoter/attenuator region of E. coli may cause constitutive
hyperexpression of campC.
2. acquire plasmid mediated Amp C

25. Need for routine laboratory detection of Amp C
Therapeutic and infection control considerations
Treatment failure Susceptible in vitro, may become resistant over a period
of time
Limited treatment options: resistance to beta lactamase inhibitors
Transferrable- Plasmid mediated Amp C

26. Laboratory detection: When to suspect?
 All “SPICE” group of organisms
Resistance to 3rd generation cephalosporins
Resistance to Cefoxitin
 I/R to beta lactam plus beta lactamase inhibitors
Exceptions :
 Serratia sensitive to ceftazidime
 Cefoxitin unreliable marker for Providentia, Morganella and Serratia
 ACC-1 is exceptional in not conferring resistance to cephamycins and is
actually cefoxitin inhibited

27. Phenotypic methods for Amp C detection
• Three dimensional extract test
• Amp C disk test
• Boronic acid disk test
• Cloxacillin disk test
• Disk approximation test

28. Three dimensional extract
Cefoxitin
Slit
Well with
enzyme extract
Flattening of zone
with indentation

29. Amp C disc test
ATCC E.coli lawn culture
Test strain on
filter paper disk

30. Boronic acid disk test
• 20 μl of 15
μg/ml
phenylboronic
acid
• >=5mm zone
size difference

31. Double disk synergy test

32. Disk approximation test

33. Multiplex polymerase chain reaction for plasmid
mediated AmpC genes
• Phenotypic tests do not differentiate between chromosomal and
plasmid mediated AmpC β-lactamases.
• Plasmid-mediated AmpC β-lactamases are most accurately
detected with the multiplex AmpC PCR test.

35. Vitek phenotype of pure Amp C
Anitibiotic Sensitivity
Ampicillin R
Amoxicillin clavulanic acid R
Piperacillin tazobactam S/I/R
Cefuroxime R
Ceftriaxone R
Cefoperazone sulbactam S/I/R
Cefepime S
Ertapenem S
Imipenem S
Meropenem S
Non beta lactams variable

36. Amp C plus ESBL
Anitibiotic Sensitivity
Ampicillin R
Amoxicillin clavulanic acid R
Piperacillin tazobactam S/I/R
Cefuroxime R
Ceftriaxone R
Cefoperazone sulbactam S/I/R
Cefepime S/R
Ertapenem S
Imipenem S
Meropenem S
Non beta lactams variable

37. AmpC hyper production plus porin loss/ efflux pump
Anitibiotic Sensitivity
Ampicillin R
Amoxicillin clavulanic acid R
Piperacillin tazobactam S/I/R
Cefuroxime R
Ceftriaxone R
Cefoperazone sulbactam S/I/R
Cefepime S/R
Ertapenem S/I/R
Imipenem S/I/R
Meropenem S/I/R
Non beta lactams variable

38. Treatment
Antibiotic Recommendations
Piperacillin tazobactam can be used for mild infections such as
uncomplicated cystitis
Cefepime Use when MIC< 4 µg/ml
Carbapenem preferred when cefepime
MIC is ≥4 µg/ml
Carbapenems
Non beta lactam agents
TMP-SMX, fluoroquinolones,
aminoglycosides, tetracyclines,
non inducer of Amp C & not a substrate for
hydrolysis, can be used based on
susceptibility and the type of infection
**Treat as per AST in organisms no expected to produce significant Amp C
enzyme Eg. Serratia marcescens, Morganella morganii, Providencia spp.,

39. Amp C beta lactamases Class C, Group 1
Chromosomal
Constitutive low level
• E.coli
• Shigella
• Enterobacter
• Citrobacter fruendii
Inducible
• S-Serratia
• P-Pseudomonas
• I- Indole positive proteae
• C- Citrobacter
• E-Enterobacter spp
• Klebsiella
pneumoniae
• Salmonella spp
• E.Coli
• Proteus mirabilis
Plasmid encoded
Summary
Amp C de- repression
Amp C induction Constitutive high level

40. • Thank You