![Page 1: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/1.jpg)
Lecture 5
Enzymatic destruction (ESBL)
Enzymatic modification (erm )
![Page 2: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/2.jpg)
Mechanisms of resistance
1. Modifying enzymes• erm
2. Degrading enzymes• ESBL
3. Target Change
4. Efflux pumps
![Page 3: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/3.jpg)
ESBL
Extendened Spectrum β-lactamases
![Page 4: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/4.jpg)
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
![Page 5: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/5.jpg)
Gram Negative Rods/Bacilli (GNR)
V. choleraeC. jejuni
Helicobacter pylori
EnterobacteriaceaePseudomonas
aeruginosa
Stenotrophomonas maltophilia
Acinetobacter spp.
Many other
(H. influenza, etc..)
![Page 6: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/6.jpg)
Enterobactericea(E. coli, Klebsiela, Enterobacter)
• Gram negative rods
• Colonize GI tract
• Clinical manifestations:– Urinary tract infections– Nosocomial pneumoniae– Bacteremia / Sepsis– Other
![Page 7: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/7.jpg)
Mechanism of resistance
β-lactamases
Enzymes that inactivate β -lactams by hydrolyzing the amide bond of the β -lactam ring.
![Page 8: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/8.jpg)
![Page 9: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/9.jpg)
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
![Page 10: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/10.jpg)
History of GNR resistance
1928
Fleming
1941
Penicillin use
1940 Penicillinase detected in
E. coli
1959
β -lactamase resistant penicillins: Methicillin
1960s
Broad spectrum/ extended spectrum
penicillins
1964
Cefalotin use
1965
Broad spectrum β –lactamases (TEM-1 in E. coli) 1983
Extended spectrum β-lactamases
1950 1960 1970 1980 1990 2000
1985
Carbapenem (Imipenem)
Early 1980s
3rd generation ceph.
Carbapenemases
TEM-1 widespread
2005
Tigecycline
ESBL outbreaks in
France
1976
β –lactamases inhibitors
![Page 11: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/11.jpg)
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
http://www.lahey.org/studies/webt.asp
![Page 12: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/12.jpg)
Types of β-lactamases
• 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)
• 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
![Page 13: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/13.jpg)
Genetic Mechanism
Transformation
Penicillinase blaZ
Plasmidtransfer
Broad spectrum b-lactamase
(blaTEM)
&
Mutation
ESBL(TEM related)
&
![Page 14: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/14.jpg)
ESBL• Confer resistance to 1st , 2nd, 3rd cef.
– 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
![Page 15: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/15.jpg)
CarbapenemasesPan-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
![Page 16: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/16.jpg)
AmpC β-lactamase
• Chromosomal
• Inducible
• Fully resistant to β-lactamase inhibitors
![Page 17: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/17.jpg)
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).
![Page 18: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/18.jpg)
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
•We are running out of treatment options!
![Page 19: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/19.jpg)
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
![Page 20: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/20.jpg)
Klebsiela resistant to 3rd generation cephalosporines (CDC)
0
2
4
6
8
10
12
14
1989
1990
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
Year
Per
cent
Res
ista
nce
![Page 21: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/21.jpg)
MDR (qnl, aminoglycoside 3rd ceph.) in Klebsiella pneumoniae in Europe (EARSS) 2005
![Page 22: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/22.jpg)
Risk factors
• Critically ill patients
• Long hospitalization (median 11-67 d)
• Invasive medical devices
• Heavy Ab treatment – 3rd generation cephalosporines– Also other: quinolones, TMP-SMX,
aminoglycosides, metronidazole
![Page 23: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/23.jpg)
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
![Page 24: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/24.jpg)
Enzymatic modification
The case of macrolides
![Page 25: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/25.jpg)
Enzymatic modification:
• Aminoglycosides– Acetyltransferases– Phosphotransferases– nucleotidyltransferases
• MLS (macrolides, lincosamides, streptogramin B)– erm (erythromycin resistance methylase) (most
common)– Other: hydrolases, esterases, glycosylases,
phosphotransferases, nucleotidyl-transferases and acetyltransferases
Mechanism animation
![Page 26: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/26.jpg)
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.
![Page 27: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/27.jpg)
Macrolide resistance
• Phenotypes of macrolide resistance:– MLSB– M
• Genotypes of macrolide resistance:– erm (erythromycin ribosomal methylase)– mef (specific macrolide effulx pump )
![Page 28: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/28.jpg)
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.
![Page 29: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/29.jpg)
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
![Page 30: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/30.jpg)
Macrolide resistance in S. pneumoniae (2001-2005) / Flemingham et al. J. Infection
![Page 31: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/31.jpg)
2000-2004
![Page 32: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/32.jpg)
PROTEKT US 2008 (2000-2004)
![Page 33: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/33.jpg)
Mac-R in S. pneumoniae in Finland / Bergman et al. 2006 AAC
![Page 34: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/34.jpg)
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
![Page 35: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/35.jpg)
Mac-R is GAS in Finland / Bergman et al. CID 2004
![Page 36: Lecture 5 Enzymatic destruction (ESBL) Enzymatic modification ( erm )](https://reader035.vdocuments.pub/reader035/viewer/2022062520/56815878550346895dc5d707/html5/thumbnails/36.jpg)
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