Antibiotic Resistance9.12.ppt

ANTIMICROBIAL RESISTANCE

9.21.12

Site of Action of antibiotics• Inhibition of nucleic acid synthesis (Rifampin; quinilones) • Inhibition of protein synthesis (Tetracyclines;

Chloramphenicol, macrolides, clindamycin, aminoglycosides, linezolid)

• Action on cell membrane (Polyenes; Polymyxin) • Interference with enzyme system (Trimethoprim,

Sulphamethoxazole) • Action on cell wall (Penicillin; cephalosporins, Vancomycin,

carbapenams)

Mechanisms of Drug Resistance• Change in drug target• Production of an enzyme that modifies or inactivates the

agent• Reduced accumulation of the agent

• Limited uptake• Active Efflux

• Loss of a pathway involved in drug activation

Mechanisms of Drug Resistance

Mechanisms of Drug Resistance

Mechanisms of Gram-Negative Bacterial

Resistance to Antibiotics Antibiotic ClassAntibiotic Class Mechanism of ResistanceMechanism of Resistance

CephalosporinsCephalosporins ESBLsESBLschromosomal cephalosporinaseschromosomal cephalosporinases

-Lactamase -Lactamase inhibitorsinhibitors

hyperproducers of hyperproducers of -lactamases-lactamasesnew new -lactamases resistant to inhibitors-lactamases resistant to inhibitorschromosomal cephalosporinaseschromosomal cephalosporinases

CarbapenemsCarbapenems porin mutationsporin mutationsefflux pump overproduction (excluding efflux pump overproduction (excluding

imipenem)imipenem)zinc metalloenzymes and other zinc metalloenzymes and other --

lactamaseslactamases

FluoroquinoloneFluoroquinoloness

alterations in DNA topoisomerasealterations in DNA topoisomeraseefflux mechanismsefflux mechanismspermeability changespermeability changes

Resistant StrainsRare

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Resistant Strains Dominant

Antimicrobial Exposure

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Selection for antimicrobial-resistant Strains

Campaign to Prevent Antimicrobial Resistance in Healthcare Settings

Target Alterations• PBPs: in cell membrane

• S. pneumoniae, MRSA• Intrinsic resistance, enterococci, gonococci, H. infl• D-Ala-D-Ala target: VRE

• VanA, VanB, VanC, VanD

• Alterations in ribosomes• Cell membrane changes

Protein Binding Proteins• Target for all B-lactams• found as both membrane-bound and cytoplasmic proteins

• all involved in the final stages of the synthesis of peptidoglycan, which is the major component of bacterial cell walls

• More common R mechanism for gram positive organisms• Gram neg access to PBP is limited by outer membrane

and thus other mechanisms supersede the binding to this target

Enzyme Production• Aminoglycoside modifying enzymes• B-lactamases:

• Four structural classes:• Class A: R of S aureus to penicillin, R of E coli to ampicillin and

cephalothin –plasmid mediated• Class B: hydrolyze carbapenmens/pens/cephs -chromosomal• Class C: chromosomal, active against cephalosporins • Class D: plamid mediatated

• ESBL: K. pneumoniae, E. coli : Derived from transfer of chromosomal genes for inducible amp C onto plasmids

B-lactamase

CefipimeIncreased stability to B-lactamase

Increased penetration into gram-positive

Ceftriaxone

B-lactame ring

-Lactamases: Overview• Large, diverse family of enzymes• Widely dispersed in gram-positive (chromosoaml and plasmid) and gram-negative pathogens (plasmid)

• Major mechanism of resistance to -lactams in gram-negative pathogens

• Wide range of activity: older enzymes hydrolyze older drugs, new derivatives have evolved for new drugs

• ESBLs• AmpC -lactamases• carbapenemases

-Lactamases• Major groups for gram-neg

• TEM-wide spread-plasmid and transposon• Enterobacteriaceae, Pseudomonas aeruginosa, Haemophilus

influenzae, and Neisseria gonorrhoeae

• SHV-1• Klebsiella pneumoniae (chromosomal) and E. coli (plasmid)

• Confer resistance to penicillins and first/second generation cephalosporins

1960TEM-1

SHV 1980sCefotaxime

TEM-2

-lactamaseExtended spectrum--lactamase

TEM, SHVCTX

ESBL-Mediated Resistance• Contain a number of mutations that allow them to

hydrolyze expanded-spectrum β-lactam antibiotics

• Derived from older antibiotic-hydrolyzing -lactamase enzymes (TEM-1, TEM-2, SHV-1)• a single amino acid substitution can give rise to new

ESBLs• Not as catalytically efficient • Inhibited by β-lactamase inhibitors • Susceptible to cefoxitin and cefotetan in vitro only

• 10%–40% of K pneumoniae, E coli express ESBLs

Rupp ME et al. Drugs. 2003;63:353–365.

CTM-X predominant mechanism

E. Coli predominant organism

Canton, Cur Opin in Micr 2006, Pages 466–475

Coresistances among the Enterobacteriaceae isolates of the different ESBL types.

Morosini M et al. Antimicrob. Agents Chemother. 2006;50:2695-2699

Amp-C

• Confer resistance cephamycins (cefotetan, cefoxitin) and oxyimino- -lactams (cefotaxime, ceftriaxone, ceftazidime)

• Chromosomal in SPACE organisms and are inducible• Poorly expressed in E. coli and is missing from

klebsiella and salmonella species

• Plasmid mediated on other gram-neg, usually not inducible

• Not susceptible to inhibitors

AmpC- vs ESBL-Mediated Resistance

• Different phenotypic characteristics• AmpC type -lactamases typically encoded on chromosome of gram-negative bacteria, can also be found on plasmids

• AmpC type -lactamases hydrolyze broad- and extended-spectrum cephalosporins

• ESBLs—NOT AmpC -lactamases—are inhibited by -lactamase inhibitors (eg, clavulanic acid)

• AmpC production is less effective on cefipime so best cephalosporin to test

New CLSI Laboratory Standards• Previously testing for ESBL was based on high MIC to

oxyimino-beta-lactam substrates (cetriaxone, cefotaxime, cefipime, cetaz) and susceptibility to inhibitors followed by a confirmatory test to detect the enzyme• Low sensitivity when mixed mechanisms at play, ie false positive

results, some attempts to overcome this with cloxacillin-containing Muller–Hinton agar, which inhibits AmpC activity

• When ESBL present susceptibility changed to resist for penicillins, cephalosporins and monobactams

• Current practice: MICs were changed• 1-3 doubling dilutions lower• No need for confirmation of enzyme• No change in reporting

Epidemiology of Plasmid AmpC Enzymes in the United States• Alvarez et al examined a sample of 752 resistant

K pneumoniae, K oxytoca, and E coli strains from 70 sites in 25 US states

• Plasmids encoding AmpC-type -lactamase were found in

• 8.5% K pneumoniae samples

• 6.9% K oxytoca samples

• 4% E coli samples

Carbapenemases• beta-lactamases with versatile hydrolytic capacities.• Ability to hydrolyze penicillins, cephalosporins,

monobactams, and carbapenems. • 2 major groups

• Metallo-b-lactamases (MBLs)• Major R in pseudomonas, acinetobacter, and enterobacter• Confer High level of R

• Serine b-lactamases• Oxacillinases or D b-lactamases (OxaA)

• Not as Diverse• Found mostly in acinetobacter• Confer only low level of hydrolytic activity therfore another R is necessary to

raise MIC• Class A carbapenemases

• Found in pseudomonas and enterobacter, but predominant type is found on a plasmid in Klebsiella

Mechanisms of Bacterial Resistance to Fluoroquinolones

• Mutations in DNA gyrase and topoisomerase• Overexpression of efflux pump system• Bacterial membrane permeability changes

Mechanisms of Antibiotic Resistance in Nonfermenters• P aeruginosa and Acinetobacter often multidrug resistant1

• Mechanisms of resistance include1,2

• production of ESBLs or AmpC -lactamases• increased efflux of antibiotic agent• decreased outer membrane permeability• DNA gyrase mutations• aminoglycoside modifying enzymes

Carbapenems: Resistance Issues• Mechanisms of resistance to carbapenems in P aeruginosa involve• loss of OprD protein (initially called D2 porin)• overproduction of efflux pump system

(MexA-MexB-OprM) • upregulation of other efflux system may be involved (cross-

resistance to fluoroquinolones)

• Resistance to meropenem depends on both

• Resistance to imipenem mainly mediated through loss of OprD

Carbapenems: Resistance Carbapenems: Resistance IssuesIssues

Outer membrane

Periplasm

Cytoplasmic membrane

D2 Porin (OprD)

Carbapenem nucleus

Ertapenem Imipenem

PBP1

PBP2

PBP3

PBP4

PBP5

Penicillin-binding proteins (PBPs)

Mutated or missingD2 porin

Courtesy of John Quinn, MD.

Mechanisms of Carbapenem Resistance: Impermeability

• OprD forms narrow transmembrane channels that are normally accessible only to carbapenems, not to other ß-lactams

• Loss of OprD porin is associated with decreased permeability of carbapenems and increased carbapenem MICs, whereas other ß-lactams remain active

Mechanisms of Carbapenem Resistance: Efflux Systems in P aeruginosa

• Upregulation of MexAB-OprM efflux system• associated with increased MICs of meropenem, not

imipenem

• Coregulation of MexE-MexF-OprN efflux system with OprD porin in P aeruginosa• upregulation of efflux associated with OprD• associated with increased MICs of fluoroquinolones as

well as carbapenems• mechanism sometimes selected by fluoroquinolones,

rarely by carbapenems

MRSA• Methicillin resistance is acquired via Mec A

• mobile chromosomal element called staphylococcal cassette chromosome (SCCmec)• SCCmec types I, II, and III and are multidrug resistant-large cassettes

• Health-care associated

• SCCmec type IV and type V not multidrug resistant• Community associated

MecA• Encodes penicillin binding protein (PBP) 2a

• Weak affinity for methicillin and all beta-lactams• Substitutes for the usual PBP 1-3 that have a high affinity for beta-

lactams

• Speculation of origination from CoNS

S. Pneumoniae

• Pencillin• Decreased affinity to PBP

• Can be overcome with high dose

• Macrolides• Genetic changes to binding target on ribosome-high

level can not be overcome =erm(B)• Efflux pump-lower level-may be overcome =mef (A)

• Clindamycin• Ribosomal methylation changing target erm(B)

S. pneumoniae• Fluoroquinilones

• Bind to either gyrase or topoisomerase or both• Resistance from mutations in gyrA or parC

• reduce binding of the drug to the site of activity • Mutations are step wise

• One mutation and R to cipro and levo• More than one needed for gemi and moxi

• Tetracyclines• Proteins are produced that package the drug into vessicles which

are extruded from the cell

Enterococcus• Intrinsic (chromosomal, naturally occurring) resistance to

• B-lactam• 10 to 1000 times more drug to inhibit an average Enterococcus than an

average Streptococcus • Due to penicillinase production and PBP5 production

• Aminogylcosides• Low level to streptocmycin and gentimicin • Synergism causes cell wall agent to become bactericidal• High level to tobramycin

Enterococcus-Intrinsic• Clindamycin-gene encoding efflux pump• TMP-SXZ-

• In vitro appears susceptible but in vitro is resistant• Can utilize preformed folic acid

• Vancomycin at low levels in some strains

Enterococcus• Genetic transfer to acquire new resistance

• One mechanism, involving pheromone-responsive plasmids, causes plasmid transfer between E. faecalis isolates at a very high frequency .

• Another mechanism involves plasmids that can transfer among a broad range of species and genera, although usually at a moderately low frequency .

• A third mechanism (conjugative transposition) involves transfer of specialized transposons at low frequency but to a very broad range of different kinds of bacteria . Conjugative transposons are relatively nonselective in their host range and are one of the few types of elements known to have crossed the gram-positive/gram-negative barrier in naturally occurring clinical isolates and to then cause resistance in these various hosts

Enterococcus• Acquired

• High level resistance to amnioglycosides• Loose synergy ability as well

• High level vancomycin resistance• Van gene clusters on transposons or plasmids

• Very old, probably initially resulted from pressor from natural glyocpeptides• Van A is the most common and confers highest level of resistance

• Variable level to linezolid• Depends on the number of mutations in the 23S rRNA