Lecture 5

Enzymatic destruction (ESBL)

Enzymatic modification (erm )

Mechanisms of resistance

1. Modifying enzymes• erm

2. Degrading enzymes• ESBL

3. Target Change

4. Efflux pumps

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

Gram Negative Rods/Bacilli (GNR)

V. choleraeC. jejuni

Helicobacter pylori

EnterobacteriaceaePseudomonas

aeruginosa

Stenotrophomonas maltophilia

Acinetobacter spp.

Many other

(H. influenza, etc..)

Enterobactericea(E. coli, Klebsiela, Enterobacter)

• Gram negative rods

• Colonize GI tract

• Clinical manifestations:– Urinary tract infections– Nosocomial pneumoniae– Bacteremia / Sepsis– Other

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

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

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– 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

Genetic Mechanism

Transformation

Penicillinase blaZ

Plasmidtransfer

Broad spectrum b-lactamase

(blaTEM)

&

Mutation

ESBL(TEM related)

&

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

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

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

•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)

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

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:

• 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

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

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