1. Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification (erm )

2. Mechanisms of resistance
Modifying enzymes
erm
Degrading enzymes
ESBL
Target Change
Efflux pumps

3. Extendened Spectrum β-lactamases
ESBL
Extendened Spectrum β-lactamases

4. Resistance in Gram negative bacteria
β-lactamases – the most important mechanism of resistance to β-lactam Ab (in Gr-).
ESBLs (Extended spectrum β-lactamases)
Carbapenemase
ESBL – worrisome in enterbactericea – e.coli, klebsiela
Carbapenemases – most worrisome if also have ESBL – no Ab to Rx with.

5. Gram Negative Rods/Bacilli (GNR)
V. cholerae
C. jejuni
Helicobacter pylori
Acinetobacter spp.
Gram Negative Rods/Bacilli (GNR)
Many other
(H. influenza, etc..)
Stenotrophomonas maltophilia
Pseudomonas aeruginosa
Enterobacteriaceae

6. Enterobactericea (E. coli, Klebsiela, Enterobacter)
Gram negative rods
Colonize GI tract
Clinical manifestations:
Urinary tract infections
Nosocomial pneumoniae
Bacteremia / Sepsis
Other
UTI, penumonia, bloodstream infections and intraabdominal infections
Community GR-N: Salmonella, Shigella.

7. Mechanism of resistance
β-lactamases
Enzymes that inactivate β -lactams by hydrolyzing the amide bond of the β -lactam ring.

9. β-lactamase inhibitors
Clavulonic acid: derived from Streptomyces clavuligerus
Little antibiotic effect in itself
Given in combination with a β -lactam Ab
Function: by binding the β -lactamase enzyme more efficiently than the actual β -lactam
Thus protect the β -lactam Ab from hydrolysis
Not efficient against cephalosporinases

10. History of GNR resistance
1965
Broad spectrum β –lactamases (TEM-1 in E. coli)
ESBL outbreaks in France
1940 Penicillinase detected in E. coli
1983 Extended spectrum β-lactamases
TEM-1 widespread
1950
1960
1970
1980
1990
2000
Carbapenemases
1941
Penicillin use
1983 first ESBL detected in Germany
1964
Cefalotin use
Early 1980s
3rd generation ceph.
1959
β -lactamase resistant penicillins: Methicillin
1985
Carbapenem (Imipenem)
1960s
Broad spectrum/ extended spectrum penicillins
1976
β –lactamases inhibitors
2005
Tigecycline
1928
Fleming

11. β-lactamases classification
Molecular class: A:
TEM
SHV
other B:
Metalloenzymes (carbapenemases) C:
Prototype: chromosomal ampC D:
OXA (oxacillin hydrolyzing enzymes)
Enzyme type (by substrate profile):
Penicillinase
Broad-spectrum
Extended Spectrum
Carbapenemase
Genetic classification:
plasmids mediated
Chromosomal

12. Types of β-lactamases β-lactamases
ESBLs
TEM related
SHV related
OXA related
CTX-M
Other
ampC β-lactamases
Resistant to β-lactamase inhibitors
chromosomal
Carbapenemases
Metallo- β-lactamases
Serine carbapenemases
β-lactamases
Penicillinase: gene blaZ , inducible, on transposon (can move between chromosome and plasmid).
Broad spectrum β-lactamases
(plasmid encoded)
TEM
SHV
OXA (mainly in pseudomonas)

13. Broad spectrum b-lactamase (blaTEM)
Genetic Mechanism
Plasmid
transfer
Broad spectrum b-lactamase (blaTEM)
&
Penicillinase blaZ
Mutation
ESBL
(TEM related)
&
Transformation

14. ESBL Confer resistance to 1st , 2nd, 3rd cef. Diversity of ESBL
Most are susceptible to β-lactamase inhibitors
Most are susceptible to 4th cef.
All are susceptible to carbapenems
Diversity of ESBL
SHV (widespread)
TEM (>100 types)
OXA
Predominantly in Pseudomonas
less susceptible to β-lactamase inhibitors
CTX-M
Probably independent evolution
Highly resistant to 3rd generation cephalosporines
initially in South America, Far East & Eastern Europe
Probably most frequent worldwide
Clonal spread has been documented
A number of TEM derivatives resistant to β-lactamase inhibitors, but are not ESBL
1st ESBL detected in Germany 1983 (SHV-2) in K. ozaenea
TEM-3 1st ESBL (initially named CTX-1) 1987 in France (K. pneumoniae)
TEM-12 UK 1982 (Klebsiella oxytoca)
Developed in an outbreak of K. oxytoca with TEM-1 in NICU, all treated with 3rd generation cephalosporines->1 patient with ESBL

15. Carbapenemases Pan-resistance
Carbapenem: “the magic bullet” very broad spectrum
Metallo-β-lactamases (class B)
Not susceptible to clavulonate
Serine-carbapenemases (class A+ D)
KPC (Klebsiela pneumonia carbapenemase)- plasmid associated

16. AmpC β-lactamase Chromosomal Inducible
Fully resistant to β-lactamase inhibitors

17. Further complicating matters:
More than one gene of β-lactamase / ESBL / ampC / carbapenemase can be carried on the same plasmid.
Genes of ESBL are carried on plasmids that usually carry additional resistant genes: frequently MDR
Laboratory diagnosis confusing:
susceptibility profiles sometimes
misleading: “hidden resistance” ->
CLSI guidelines are changing.
CTX-M clones appearing in the
community (Canada, Greece, Spain,
Italy).

18. Treatment of Gram negative infections:
Penicillins
Cephalosporines (1st, 2nd)
Extended spectrum Cephalosporines (3rd, 4th)
Quinolones
β-lactam-β-lactamase inhibitors
Carbapenems
Colistin…Tigecycline
β-lactamase (penicillinase)
Broad spectrum β -lactamase
ESBL
Quinolone resistance
ESBL (OXA)
ampC
Carbapenemases
3rd generation was developed to overcome resistance to 1st/2nd generations.
Within a few years of introduction – developed mutants and the b-lactamases became ESBLs
ESBL usually also resistant to aminoglycosides, Trimethoprim-sulfametoxazole, and quinolones
B-lactam inhibitors: Pipracillin-tazobacam, amoxycillin-clavulonate, ampicllin-sulbactam
We are running out of treatment options!

19. The evolution of ESBL In a single patient:
SHV-1-> 3rd Cef Rx. -> SHV-8
ESBL TEM-24 from:
Enterobacter aerogenes -> E. coli -> proteus mirabilis -> Pseudomonas aeruginosa
Mutations + efficient horizontal transmission
K. pneumoniae the major ESBL producer

20. Klebsiela resistant to 3rd generation cephalosporines (CDC)

21. MDR (qnl, aminoglycoside 3rd ceph
MDR (qnl, aminoglycoside 3rd ceph.) in Klebsiella pneumoniae in Europe (EARSS) 2005

22. Risk factors Critically ill patients
Long hospitalization (median d)
Invasive medical devices
Heavy Ab treatment
3rd generation cephalosporines
Also other: quinolones, TMP-SMX, aminoglycosides, metronidazole

23. Control of ESBL outbreaks
Monoclonal
Indicates transmission from patient to patient.
Probably induced by lack of IC measures
Infection Control
Polyclonal
Indicates multiple events of evolving resistance.
Probably induced by selective Ab pressure
Antibiotic control

24. Enzymatic modification
The case of macrolides

25. Enzymatic modification:
Mechanism animation
Aminoglycosides
Acetyltransferases
Phosphotransferases
nucleotidyltransferases
MLS (macrolides, lincosamides, streptogramin B)
erm (erythromycin resistance methylase) (most common)
Other: hydrolases, esterases, glycosylases, phosphotransferases, nucleotidyl-transferases and acetyltransferases

26. Macrolide resistance
Macrolides are used to treat Gram+ bacteria and atypical bacteria (mycoplasma, legionella, chlamidia).
Bacteriostatic
Macrolides act by inhibiting protein synthesis, by binding to 50S subunit of the ribosome of the bacteria.

27. Macrolide resistance Phenotypes of macrolide resistance:
MLSB M
Genotypes of macrolide resistance:
erm (erythromycin ribosomal methylase)
mef (specific macrolide effulx pump )

28. erm Erythromycin ribosomal methylase:
The predominant macrolide resistance mechanism.
34 different classes of Erm proteins.
Each functions by methylating a single adenine residue of the 23S rRNA.
Methylation results in MLSB pheontype (resistance to most macrolides).
Can be either inducible or constitutive.

29. Macrolide resistance in S. pneumoniae
ermB
predominant in most of the world
High level resistance (MIC>64)
mefA
most common in some areas (USA)
low level resistance (MIC 4-8)
Increasing level of resistance
Changing epidemiology
Strains containing both mefA + ermB emerging (from 10% to 18% in last 4 y)
mefA + ermB usually clonally related to MDR (19A – non-vaccine type)
Correlation between increasing consumption of mac and Mac R in SP
Teilithromycin (Ketolide) effective against mefA +ermB R.

30. Macrolide resistance in S. pneumoniae (2001-2005) / Flemingham et al. J. Infection

32. PROTEKT US 2008 ( )

33. Mac-R in S. pneumoniae in Finland / Bergman et al. 2006 AAC

34. Macrolide resistance in GAS
Uncommon: US<5%
Single outbreak in Pittsburg (up to 48% Mac-R, single clone)
Mechanisms:
ermA (ErmA subclass TR)
ermB
mefA
All associated with mobile genetic elements

35. Mac-R is GAS in Finland / Bergman et al. CID 2004

36. Macrolide R in S. aureus
Clindamycin resistance – an important treatment issue.
Mechanism of resistance:
Target modification (MLSBi) (ermA, ermC)
Efflux pumps (MS phenotype:
not clinda R) (msrA)
Inactivation
ermB in staph, not aureus and not human.