1. COL Helen Viscount, PhD, D(ABMM) LTC Steven Mahlen, PhD, D(ABMM)
The Beta-Lactamase Family: Classification, Detection, and Interpretive Criteria
COL Helen Viscount, PhD, D(ABMM)
LTC Steven Mahlen, PhD, D(ABMM)

2. Transplant patient Extremely resistant Klebsiella pneumoniae recovered
Sensitive only to colistin and gentamicin
Patient put in isolation
Isolate transmitted to 10 other patients
Outcomes:
4/5 with bacteremia died
1 other died
2 with renal failure
Only 4/11 discharged without renal failure
Ampicillin: R
Pip/tazo: R
Ceftazidime: R
Ceftriaxone: R
Cefepime: R
Imipenem: R
Meropenem: R
Aztreonam: R
Amikacin: R
Tobramycin: R
Trimeth/sulfa: R
Fluoroquinolones: R
Gentamicin: S
Colistin: S

3. Nursing home resident 83 years old Pneumonia Blood cultures +
Admitted to ICU
Started on ceftriaxone and levofloxacin
Blood cultures +
K. pneumoniae
Based on sensi’s:
No more levo
Kept on ceftriaxone
Patient got worse
Had to be ventilated
Ampicillin: R
Pip/tazo: S
Cefazolin: R
Ceftazidime: I
Ceftriaxone: S
Cefepime: S
Imipenem: S
Aztreonam: S
Tobramycin: S
Trimeth/sulfa: R
Levofloxacin: I
Ciprofloxacin: I
Gentamicin: S

4. Objectives
At the end of this workshop the attendee should be able to distinguish ESBL positive from carbapenemase- producing bacteria
At the end of this workshop the attendee should be able to describe a method to screen for ESBLs
At the end of this workshop the attendee should be able to interpret the results of the modified Hodge Test

5. Beta-lactam antibiotics
Penicillins
Ampicillin
Amoxicillin
Piperacillin
Cephalosporins (generations)
1st gen: cephalothin
2nd gen (cephamycins): cefoxitin, cefotetan
3rd gen: ceftazidime, cefotaxime, ceftriaxone
4th gen: cefepime

6. Beta-lactam antibiotics
Monobactam: aztreonam
Carbapenems:
Imipenem
Meropenem
Ertapenem
Inhibitors
Sulbactam (ampicillin/sulbactam: Unasyn)
Tazobactam (piperacillin/tazobactam: Zosyn)
Clavulanate (amoxicillin/clavulanate: Augmentin)

7. Mechanisms of Resistance
Altered target (Gram negative/positive)
Altered permeability (Gram negative)
Production of inactivating enzymes (Gram negative/positive)

8. Gram-negative cell Gram-positive cell Outer membrane Peptidoglycan
Penicillin
Binding proteins
(PBPs)
Inner (cytoplasmic) membrane

9. Alteration of Target
Resistance to -lactams via altered penicillin-binding proteins (PBPs)
MRSA
Vancomycin resistance in enterococci
Fluoroquinolone resistance

10. Altered Permeability Passive diffusion of Gram-negative cell wall
Mutate outer membrane proteins
Active efflux

11. Active Efflux

12. Production of Inactivating Enzymes
Chloramphenicol acetyltransferase
Aminoglycoside-modifying enzymes
-Lactamases

13. -Lactamases Well over 340 different enzymes
Extended spectrum -lactamases (ESBLs)
AmpC -lactamases
Chromosomal
Plasmid-mediated
Carbapenemases

14. -Lactamases First -lactamase identified: AmpC beta-lactamase
1940, Escherichia coli
1940, penicillinase, Staphylococcus aureus
First plasmid-mediated -lactamase: TEM-1
1965, Escherichia coli, Greece

15. -Lactamase Activity C N H
R-CONH S
COOH
CH3 O
-lactam
Enzyme-Ser-OH

16. -Lactamase Activity H H S R-CONH C C CH3 O C N CH3 O H COOH HOH Ser
Enzyme

17. L    L     L         L    L  L     L  
b-lactamase
production  L  L 

18. Types of Beta-Lactamases
ESBLs
AmpCs
Carbapenemases

19. ESBLs

20. ESBLs
Extended-spectrum beta-lactamases (ESBLs) are mutant enzymes with a broader range of activity than their parent molecules
They:
Hydrolyze 3rd and 4th gen cephalosporins and aztreonam
Do not affect cephamycins (2nd gen ceph) or carbapenems
Remain susceptible to beta-lactamase inhibitors

21. ESBLs
The most common plasmid-mediated ß-lactamases in Enterobacteriaceae are TEM-1, TEM-2, and SHV-1
TEM: Escherichia coli
Named after first patient with a urinary tract infection that was not treatable with ampicillin
Her name: Temorina
SHV: Klebsiella pneumoniae
“Sulfhydryl variant”; amino acids in the enzyme that cross-link with other molecules
“Classical” ESBLs are derived from TEM and SHV enzymes
“Non-classical” ESBLs are derived from enzymes other than TEM or SHV

22. Classical ESBLs Primarily found in E. coli and Klebsiella spp.
Differ from their parent TEM or SHV enzymes by only 1-4 amino acids
>100 TEM- or SHV-derived beta-lactamases have been described – most are ESBLs

23. Non-classical ESBLs
Many described, but less common than classical ESBLs
CTX-M
Found in multiple genera of Enterobacteriaceae
Preferentially hydrolyze cefotaxime
U.S., Europe, South America, Japan, Canada
OXA
Mainly in P. aeruginosa
Primarily hydrolyze ceftazidime
France, Turkey

24. ESBL Epidemiology ESBLs first appeared in Europe in the mid-1980s
Worldwide, but prevalence varies widely geographically and between institutions
U.S. national average for ESBLs in Enterobacteriaceae ~3%

25. ESBL Epidemiology
ESBL producers especially prevalent in ICUs and long term care facilities
Becoming more widespread in the community also
Have been associated with outbreaks
Typically arise in ICU
Plasmid transfer between GNRs
Organism transfer between patients
Control of outbreaks
Infection control practice – isolation
Restriction of 3rd and 4th generation cephalosporins
Antimicrobial cycling

26. Clinical Significance
Despite appearing susceptible to one or more penicillins, cephalosporins, or aztreonam in vitro, the use of these agents to treat infections due to ESBL-producers has been associated with poor clinical outcome

27. Clinical Significance
ESBL genes are often carried on plasmids that also encode resistance to multiple classes of antimicrobials
Aminoglycosides, Fluoroquinolones
Trimethoprim/Sulfamethoxazole
Treatment experience is largely based on classical ESBL producers
Carbapenems
ß-lactam/inhibitor combinations

28. Typical ESBL Susceptibility Profile
Amp: R
Piperacillin: R
Pip/tazo: S
Cefazolin: R
Cefoxitin: S
Ceftazidime: S
Ceftriaxone: R
Cefepime: R
Aztreonam: S
Imipenem/meropenem: S
Amp: R
Piperacillin: R
Pip/tazo: S
Cefazolin: R
Cefoxitin: S
Ceftazidime: R
Ceftriaxone: R
Cefepime: R
Aztreonam: R
Imipenem/meropenem: S

29. AmpCs

30. AmpC: General Chromosomal Escherichia coli Citrobacter freundii
Enterobacter aerogenes, E. cloacae
Serratia marcescens
Morganella morganii
Hafnia alvei
Providencia rettgeri, P. stuartii
Pseudomonas aeruginosa
Aeromonas sp.

31. AmpC: General Are not inhibited by -lactamase inhibitors
Normally are repressed, so produced at low levels
Chromosomal: inducible
In all except E. coli
In the presence of certain -lactam antibiotics
Normally, produced at low levels
Plasmid-mediated also

32. The AmpC of E. coli Chromosomal, but not inducible
Normally expressed at low levels
Regulated by a growth rate-dependent attenuation mechanism
Can become highly expressed with mutations
Amp: S
Amox/clav: S
Piperacillin: S
Pip/tazo: S
Cefoxitin: S
Ceftazidime: S
Ceftriaxone: S
Cefepime: S
Aztreonam: S
Imipenem/meropenem: S

33. AmpC Induction and Derepression
Is induction clinically relevant?
True danger—mutation in induction pathway
“Derepressed mutant”
fold more enzyme produced than normal

34. Chromosomal AmpC profile
Normal
Amp: R
Amox/clav: R
Piperacillin: S
Pip/tazo: S
Cefoxitin: R
Ceftazidime: S
Ceftriaxone: S
Cefepime: S
Aztreonam: S
Imipenem/meropenem: S
Derepressed profile
Amp: R
Amox/clav: R
Piperacillin: R
Pip/tazo: R
Cefoxitin: R
Ceftazidime: R
Ceftriaxone: R
Cefepime: S
Aztreonam: R
Imipenem/meropenem: S

35. Plasmid-Mediated AmpCs (pAmpC)
First true proof of AmpC on plasmid: 1988
MIR-1, found in Klebsiella pneumoniae
90% identical to E. cloacae ampC
Some are also inducible (DHA-1)
Most frequently found in K. pneumoniae
Also commonly found in:
K. oxytoca
Salmonella sp.
P. mirabilis
E. coli, E. aerogenes also

36. pAmpCs: Distribution World-wide distribution
Africa, Asia, Europe, Middle East, North America, South America, Central America
CMY-2 is most prevalent globally
Algeria, France, Germany, Greece, India, Pakistan, Taiwan, Turkey, UK, US

37. ESBLs vs AmpCs ESBLs AmpCs S R Cefoxitin, cefotetan
Inhibitors (pip/tazo, amp/sulbactam, amox/clav) S R
Cefoxitin, cefotetan
Ceftazidime, ceftriaxone
Cefepime
S/R

38. Carbapenemases

39. Carbapenemases Carbapenem resistance:
High level production of chromosomal AmpC with decreased outer membrane permeability (porins)
E. cloacae, E. aerogenes
C. freundii
E. coli
S. marcescens
K. pneumoniae (porins)

40. Carbapenemases Carbapenem resistance:
Changes in affinity of PBPs for carbapenems
Carbapenemases
Frequently, bugs that produce a carbapenemase produce other -lactamases

41. Carbapenemases KPC (plasmid, K. pneumoniae)
“Klebsiella pneumoniae carbapenemase”
IMI-1 (plasmid, E. cloacae)
Nmc-A (plasmid, E. cloacae)
Sme-1 (plasmid S. marcescens)
IMP-1 (plasmid, S. marcescens, P. aeruginosa)
L-1 (chromosomal, Stenotrophomonas maltophilia)

42. Carbapenemases: Profile
R to carbapenems, penicillins, cephalosporins
S or R to aztreonam, depending on enzyme
So the key:
Look for intermediate or R to imipenem or meropenem!

43. KPC Infection control emergency!!!
May test sensitive to carbapenems though!
Extensive multidrug resistance (XDR)
Very rapid spread
Empiric therapy: colistin + tigecycline
KPC 1-8

44. Further reading Yang, 2007. Ann. Pharmocother. 41:1427-1435
Jacoby, Clin. Microbiol. Rev. 22:
Black et al, J. Clin. Microbiol. 43:
Livermore et al, J. Antimicrob. Chemother. 48 Suppl 1:
Pfaller and Segreti, Clin. Infect. Dis. 42: S