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

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

Extendened Spectrum β-lactamases ESBL Extendened Spectrum β-lactamases

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.

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

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.

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

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

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

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

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)

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

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

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

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

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).

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!

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

Klebsiela resistant to 3rd generation cephalosporines (CDC)

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

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

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

Enzymatic modification The case of macrolides

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

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.

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

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.

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.

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

2000-2004

PROTEKT US 2008 (2000-2004)

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

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

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

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.