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

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

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

3. 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

5. 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

7. β-lactamases Mechanism of resistance 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. 1950 1960 1970 1980 1990 2000 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 Carbapenemases 1964 Cefalotin use 1941 Penicillin 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. 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 β-lactamases classification http://www.lahey.org/studies/webt.asp

12. 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 ampCβ-lactamases Resistant to β-lactamase inhibitors chromosomal Carbapenemases Metallo- β-lactamases Serine carbapenemases Types of β-lactamases

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

14. 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

15. 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

16. AmpCβ-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. 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 Treatment of Gram negative infections: • 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.) in Klebsiella pneumoniae in Europe (EARSS) 2005

22. 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

23. 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 Control of ESBL outbreaks

24. Enzymatic modification The case of macrolides

25. Mechanism animation 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

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. ermErythromycin 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 inS. 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

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

31. 2000-2004

32. PROTEKT US 2008 (2000-2004)

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