ANTIMICROBIAL RESISTANCE

Dr. Areej MuftiMD, FRCPC, ABMM, FCCMConsultant Microbiology- KAMC;WRAssist. Professor- KSUHS

CDC REPORT CASE.. 41 years old lady admitted with aplastic

anemia and fever. Started on cefotaxime

Week 1:Blood cultures + E.coli isolates R TO amp/ narrow spectrum

cephalosporines. Week2-4: persistent fever and bacterimia

? Other suspected organism..no, only EC Varieties of antimicrobial: genta, ticarcillin,

cefotaxime, ceftazidime, vanco, clinda.. Patient was not improved…micro lab

contacted..AJIC Tenover et al, 2006

QUESTIONS IN CONCERNS

Why is antimicrobial resistance a concern?

How do antimicrobial agents work?

How to detect Antimicrobial susceptibility

Mechanisms of resistance to antimicrobial agents(some case scenarios)

WHY IS ANTIMICROBIAL RESISTANCE A CONCERN?

•Approximately one quarter of the PubMed citations for “NOSOCOMIAL ACINETOBACTER” in the past 20 years appeared in years 2005-2006.

•At > 300 US hospitals, carbapenems R- rates increased from 9% in year 1995 to 40% in 2004( CDC survey)

NATURE MEDICINE:OPINION: ANTIBIOTIC BILL DOESN’T GAIN ENOUGH GROUND

The prevalence of hospital-acquired superbugs has reached an all-time high, claiming the lives of as many as 70,000 people per year.

08 Jul 2011By Paul G. Ambrose

WHY IS RESISTANCE A CONCERN? More treatment failures..

More mortality rate.

More financial cost.

More toxicities from antimicrobials exposure.

Prolonged therapy may lead to the development of low level resistance that:

a. compromise the therapy. b. may not be detected by routine susceptibility

testing methods used in hospital labs.

SINCE YEAR 2001…..

CASE SCENARIO… 54 years old male diagnosed with

diabetic foot.

swabs from the wound revealed polymicrobial causing organisms including SA, Kleb. pne.

Decision was made to start daptomycin..

HOW DOES DAPTOMYCIN WORK?

a. Interference with cell wall.

b. Protein synthesis inhibition.

c. Interference with nucleic acid synthesis.

d. Inhibition of metabolic pathway

e. Disruption of bacterial membrane structure

QUESTIONS IN CONCERNS

Why is resistance a concern?

How do antimicrobial agents work?

How to detect Antimicrobial susceptibility

Mechanisms of resistance to antimicrobial agents(some case scenarios)

EITHER:PHENOTYPIC OR GENOTYPIC

How to detect Antimicrobial susceptibility

ANTIMICROBIAL SUSCEPTIBILITY TESTS Minimum inhibitory concentration [MIC]

The smallest concentration of antibiotic that inhibits the growth of organism.

Liquid media (dilution) allows MIC estimation

Solid media (diffusion) Disk diffusion (Kirby-Bauer) E-tests Allows MIC estimation

Beta lactamase production: quick screening method

KIRBY-BAUER DISC TESTINGAntibiotic-impregnated discs placed on an agar plate at

theinterface between test organism and susceptible control

organismResulting zones of inhibition compared, use of controls Susceptibility is inferred (standard tables)

DILUTION IN LIQUID BROTH Tubes containing increasing antibiotic

concentrations Incubation during 18 hr at 37°C Tedious

0 (Control) 0,25 0,50 1 2 4 8 mg/l

MICBacterial growth Inhibition

READING E-TESTS

Susceptible < 1

Resistant > 4 ug/ml

Ciprofloxacin for Yersinia pestis

Intermediate 1-4 ug/ml

Upper reading

ESBL DETECTION

Wu T et al .2001;47:755-761

MODIFIED HODGE TEST FOR CARBAPENEMASE DETECTION IN ENTEROBACTERIACEAE

Lawn of carbapenem S strain: E. coli ATCC 25922 is made, place ertapenem disc, then make a streak of testing organism.

If the isolate produce carbapenemase, it will allow growth of carabapenem sensetive EC towards carbapenem disc=cloverleave like indentation.

Limitations:

•The class of carbapenemase cannot be determined by this test.

•Some isolates show slight indentation but don’t produce carbapenemase.

•MAINLY FOR EPIDMIOLOGICAL STUDY. CDC

STAPHYLOCOCCUS AUREUS AND INDUCIBLE RESISTANCE TO CLINDAMYCIN

Test for inducible resistance to clindamycin using D test in erythromycin resistant isolates

Methylation of an adenine residue of bacterial 23S rRNA (MLSB phenotype, ermB)

Effective in treatment of CA-MRSA in the absence of inducible resistance

Clin Infect Dis 2003;37:1257-60

Pediatr Infect Dis J 2003;22:593-8Pediatr Infect Dis J 2002;21:530-4

MAKE A DECISION 12 years old boy, diabetic, quadriplegic secondary to car

accident 2 years ago, on permanent urinary catheter, noticed to have turbid foul smelling urine. You saw him as an outpatient.

Urine culture was sent: + for pseudomonas with the following sensitivity profile:

-Ceftazidime: S - Ciprofloxacin: R -Gentamicin: S - trimethoprim- sulfamethoxazole: S - Nitrofurantoin: S

WHAT IS YOUR DECISION….?

QUESTIONS IN CONCERNS

Why is antimicrobial resistance a concern?

How do antimicrobial agents work?

How to detect Antimicrobial susceptibility

Mechanisms of resistance to antimicrobial agents(some case scenarios)

MECHANISMS OF RESISTANCE TO ANTIMICROBIAL AGENTS

Natural resistance Chromosomic

genetic support.

Affect almost all species strains.

Existed before antibiotic use (Enterobacter sp. - amoxicillin)

Acquired resistance Chromosomic,

plasmidic or transposon genetic support.

Affects a fraction of strains.

Increased with antibiotic use(extended spectrum beta-lactamase producing E. coli)

NATURAL RESISTANCE

ORGANISMS NATURAL RESISTANCE AGAINST:

MECHANISM

Gram-positive bacteria Aztreonam Lack of penicillin binding proteins (PBPs) that bind and are inhibited by this beta lactam antibiotic

Gram-negative bacteria Vancomycin Lack of uptake resulting from inability of vancomycin to penetrate outer membrane

Klebsiella spp. Ampicillin Production of beta-lactamases that destroy ampicillin before the drug can reach the PBP targets

Stenotrophomonas. maltophila

Imipenem Production of beta lactamases that destroy imipenem before the drug can reach the PBP targets.

ORGANISMS NATURAL RESISTANCE AGAINST:

MECHANISM

Anaerobic bacteria Aminoglycosides Lack of oxidative metabolism to drive uptake of aminoglycosides

Lactobacilli and Leuconostoc Vancomycin Lack of appropriate cell wall precursor target to allow vancomycin to bind and inhibit cell wall synthesis

Pseudomonas aeruginosa Sulfonamides, trimethoprim, tetracycline, or chloramphenicol

Lack of uptake resulting from inability of antibiotics to achieve effective intracellular concentrations

Enterococci

Aminoglycosides Lack of sufficient oxidative metabolism to drive uptake of aminoglycosides

Enterococci All cephalosporins Lack of PBPs that effectively bind and are inhibited by these beta lactam antibiotics

Aerobic bacteria Metronidazole Inability to anaerobically reduce drug to its active form

ACQUIRED RESISTANCE:EITHER

Genetic exchange : Horizontal evolution.

SPONTINOUS Mutation or selection :Vertical evolution.

HORIZONTAL EVOLUTION: acquisition of new

resistant genetic material from other R organisms.

May occur between strains of same species or between different bacterial species or genera.

Usually >> MDR organism.

(pilus)

HORIZONTAL EVOLUTION

VRSA from VRE, year 2002, NY

Transposon: segment of DNA that is

capable of independently replicating itself and inserting the copy into a new position within the same or another chromosome or plasmid.

PlasmidA circular extrachromosomal

genetic element that replicates within a cell independently of the chromosomal DNA

ACQUIRED RESISTANCE

Altered target (Gram negative/positive)

Altered permeability (Gram negative)

Production of inactivating enzymes (Gram negative/positive)

Gram-negative cell Gram-positive cell

Outer membrane

PeptidoglycanPeptidoglycan

PenicillinBinding proteins(PBPs)

Inner (cytoplasmic) membrane

ALTERATION OF TARGET Resistance to -lactams via altered

penicillin-binding proteins (PBPs):

MRSA.

Pen-R S. pneumoniae

ALTERED PERMEABILITY down regulation or altered an outer

membrane protein( porin) channel that the drug requires for cell entry:

passive diffusion of Gram-negative cell wall as OmF in E.coli: cephamycins.

Up regulating pumps that expel the drug from the cell: Active efflux e.g. Cipro against SA

PRODUCTION OF INACTIVATING ENZYMES Chloramphenicol acetyltransferase

Erythromycin ribosomal methylase:SA

Aminoglycoside-modifying enzymes.

-Lactamases

MECHANISMS OF RESISTANCE TO ANTIMICROBIAL AGENTS

BETA-LACTAM ANTIBIOTICS Penicillins

AmpicillinAmoxicillinPiperacillin

Cephalosporins (generations)1st gen: cephalothin2nd gen (cephamycins): cefoxitin, cefotetan3rd gen: ceftazidime, cefotaxime,

ceftriaxone4th gen: cefepime5TH gen:

BETA-LACTAM ANTIBIOTICS Monobactam: aztreonam Carbapenems:

ImipenemMeropenemErtapenem

InhibitorsSulbactam (ampicillin/sulbactam: Unasyn)Tazobactam (piperacillin/tazobactam:

Zosyn)Clavulanate (amoxicillin/clavulanate:

Augmentin)

EXTENDED-SPECTRUM BETA-LACTAMASES (ESBL) Definition.

Varieties.

Laboratory detection..

EXTENDED-SPECTRUM BETA-LACTAMASES (ESBL): DEFINITION enzymes that confer resistance to most

beta-lactam antibiotics, including penicillins, cephalosporins(3rd/4th), and the monobactam aztreonam. Do not affect cephamycins (2nd gen ceph) or

carbapenems.

Remain susceptible to beta-lactamase inhibitors

( in vitro)

EXTENDED-SPECTRUM BETA-LACTAMASES (ESBL): HISTORY First -lactamase identified: AmpC beta-

lactamase 1940, Escherichia coli 1940, penicillinase, Staphylococcus aureus.

First plasmid-mediated -lactamase: TEM-1 1965, Escherichia coli, Greece

1. . It was named TEM after the patient from whom it was isolated :UTI not treated with Ampicillin(Temoniera, Greece).

2. Subsequently, a closely related enzyme was discovered and named TEM-2( differs from TEM-1) by a single amino acid.

The most common plasmid-mediated ß-lactamases in Enterobacteriaceae are TEM-1, TEM-2, and SHV-1

SHV: Klebsiella pneumoniae “Sulfhydryl variant”; amino acids in the enzyme

that cross-link with other molecules “Classical” ESBLs are derived from TEM

and SHV enzymes “Non-classical” ESBLs are derived from

enzymes other than TEM or SHV

CLASSICAL ESBLS Primarily found in E. coli and Klebsiella

spp. Differ from their parent TEM or SHV

enzymes by only 1-4 amino acids >100 TEM- or SHV-derived beta-

lactamases have been described – most are ESBLs

NON-CLASSICAL ESBLS Many described, but less common

than classical ESBLsCTX-M

Found in multiple genera of Enterobacteriaceae Preferentially hydrolyze cefotaxime U.S., Europe, South America, Japan, Canada

OXA Mainly in P. aeruginosa Primarily hydrolyze ceftazidime France, Turkey

CLINICAL SIGNIFICANCE

Despite appearing susceptible to one or more penicillins, cephalosporins, or aztreonam in vitro, the use of these agents to treat infections due to ESBL-producers has been associated with poor clinical outcome

CLINICAL SIGNIFICANCE ESBL genes are often carried on

plasmids that also encode resistance to multiple classes of antimicrobialsAminoglycosides, FluoroquinolonesTrimethoprim/Sulfamethoxazole

Treatment experience is largely based on classical ESBL producersCarbapenemsß-lactam/inhibitor combinations

-LACTAMASE ACTIVITY

C C

C N

H H

R-CONHS

COOH

CH3

CH3

O

Enzyme-Ser-OH

-lactam

-LACTAMASE ACTIVITY

C C

C N

H H

R-CONHS

COOH

CH3

CH3

O

HO

Ser

Enzyme

HOH

TYPES OF BETA-LACTAMASES Well over 340 different enzymes.

Extended spectrum -lactamases:ESBLs AmpC -lactamases :

ChromosomalPlasmid-mediated

Carbapenemases

AMPC: GENERAL Chromosomal

Escherichia coli Citrobacter freundii Enterobacter aerogenes,

E. cloacae Serratia marcescens Morganella morganii Hafnia alvei Providencia rettgeri, P.

stuartii Pseudomonas aeruginosa Aeromonas sp.

AMPC: GENERAL Are not inhibited by -lactamase

inhibitors.

Normally are repressed, so produced at low levels.

Chromosomal: inducible In the presence of certain -lactam

antibioticsNormally, produced at low levels.

Plasmid-mediated also reported.

CHROMOSOMAL AMPC PROFILE Normal

Amp: R Amox/clav: R Piperacillin: S Pip/tazo: S Cefoxitin: R Ceftazidime: S Ceftriaxone: S Cefepime: S Aztreonam: S Imipenem/

meropenem: S

Derepressed profile Amp: R Amox/clav: R Piperacillin: R Pip/tazo: R Cefoxitin: R Ceftazidime: R Ceftriaxone: R Cefepime: S Aztreonam: R Imipenem/

meropenem: S

PLASMID-MEDIATED AMPCS (PAMPC) First true proof of AmpC on plasmid:

1988MIR-1, found in Klebsiella pneumoniae90% identical to E. cloacae ampC

Some are also inducible (DHA-1) Most frequently found in K. pneumoniae Also commonly found in:

K. oxytocaSalmonella sp.P. mirabilis

E. coli, E. aerogenes also.

AMPC INDUCTION AND DEREPRESSION

Is induction clinically relevant?

True danger—mutation in induction pathway:

“Derepressed mutant”

150-1000 fold more enzyme produced than normal

35 Y O WOMAN DEVELOPS A CATHETER-RELATED BLOODSTREAM INFECTION WITH ENTEROBACTER.

MONOTHERAPY WITH WHICH OF THE FOLLOWING ANTIBIOTICS WOULD BE LEAST PREFERRED EVEN THOUGH THE ORGANISM IS SUSCEPTIBLE TO ALL THREE?

A. CeftazidimeB. CefepimeC. Imipenem

ESBLS VS AMPCS

ESBLs AmpCsInhibitors (pip/tazo, amp/sulbactam, amox/clav)

S RCefoxitin, cefotetan S RCeftazidime, ceftriaxone R R

Cefepime S/R S

ESBL AmpCBugs E. coli, Klebsiella SPICEM organisms

(Serratia, Pseudomonas, Providencia, Indole-pos Proteus, Citrobacter, Enterobacter, Morganella

Genetics Plasmid Chromosome or plasmid

Inducible Resistance No Yes*

Most stable β-lactams Carbapenem Carbapenem or cefepime

*Monotherapy with penicillin or 3rd generation cephalosporin may be associated with inducible resistance

PROBLEMATIC Β-LACTAMASES

CARBAPENEMASES

Carbapenem resistance:Changes in affinity of PBPs for carbapenemsCarbapenemases.

Frequently, bugs that produce a carbapenemase produce other -lactamases

CARBAPENEMASES KPC (plasmid, K. pneumoniae)

“Klebsiella pneumoniae carbapenemase” IMI-1 (plasmid, E. cloacae) Nmc-A (plasmid, E. cloacae) Sme-1 (plasmid S. marcescens) IMP-1 (plasmid, S. marcescens, P.

aeruginosa) L-1 (chromosomal,

Stenotrophomonas maltophilia)

KPC Infection control emergency!!!

May test sensitive to carbapenems though! Extensive multidrug resistance (XDR) Very rapid spread Empiric therapy: colistin + tigecycline KPC 1-8

CARBAPENEMASES: PROFILE

R to carbapenems, penicillins, cephalosporins.

S or R to aztreonam, depending on enzyme.

So the key:Look for S with high MIC, intermediate or R

to imipenem or meropenem!

MACROLIDE RESISTANCE

Efflux of drug in S. pyogenes, S. pneumoniaeM phenotype encoded by mef gene

Alteration of 23S rRNA of 50S ribosomal subunit by methylation of adenineAssociated with resistance to macrolides,

lincosamides (clindamycin), and streptogramin type B (MLSB phenotype)

ermB gene

WHICH OF THE FOLLOWING IS CORRECT REGARDING S. PNEUMONIAE?

A. The MIC susceptibility breakpoint for penicillin has recently been lowered due to increased clinical failure with penicillin treatment.

B. Levofloxacin is the most active fluoroquinolone against S. pneumoniae

C. The addition of a beta-lactamase inhibitor (ampicillin-sulbactam) can overcome the penicillin resistance.

D. Introduction of the pneumococcal conjugate vaccine has been associated with a reduction in non-penicillin susceptible invasive pneumococcal infections.

PNEUMOCOCCAL CONJUGATE VACCINE

Rate of penicillin-nonsusceptible invasive disease per 100,000

1999 2004All ages 6.3

2.7Children < 2 years of age 70.3 13.1Persons > 65 years of age 16.4 8.4

Serotype 19A 0.3 1.2 Children < 2 years of age 0.8 8.3

Meningitis per 100,000 1994-1999 2001-2004

Children < 2 years of age 7.7 2.6 Persons > 65 years of age 1.2 0.8

N Engl J Med 2006;354:1455-63

Clin Infect Dis 2008;46:1664-72

S. PNEUMONIAE Β-LACTAM RESISTANCE

Clin Infect Dis 2009;48:1596-1600SENTRY surveillance: susceptibility increase from 68% to 93% of isolates

Ceftriaxone Susceptible Intermediate Resistant

Previous < 0.5 1.0 > 2.0

Non-meningitis <1.0 2.0 > 4.0

TETRACYCLINES tet efflux genes Tigecycline is a new glycylcycline

derivative of minocyclineDesigned to overcome drug-resistance due to

efflux and ribosomal protection In vitro activity against PRSP, MRSA, VRE, and

some Acinetobacter, but not PseudomonasEmergence of resistance on therapy,

particularly with Acinetobacter

WHICH OF THE FOLLOWING IS CORRECT REGARDING S. AUREUS RESISTANCE?

A. Daptomycin is active against VISA, but not VRSAB. VRSA isolates to date have contained vanBC. Breakpoint for vancomycin susceptibility is 4.0

mcg/mlD. MecA gene encodes PBP2aE. Isolates susceptible to erythromycin should

undergo “D-test” for inducible clindamycin resistance

F. Linezolid resistance is due to drug efflux

VANCOMYCIN RESISTANCE Binds to cell wall precursors ending

in D-Ala-D-Ala and prevents their incorporation into cell wall synthesis

Vancomycin-intermediate resistant S. aureus (VISA) First documented in Japan 1996, US in

1997 Increased cell wall thickness limiting

glycopeptide access to site of cell wall synthesis

Vancomycin-resistant S. aureus (VRSA) Isolated in June 2002 Contained vanA resistance gene identical

to vanA gene in patient’s vancomycin-resistant Enterococcus faecalis

van genes encode for precursors with alternative termini that have low affinity for vancomycin (eg. vanA encodes D-Ala-D-Lac)

VISA

REDUCED SUSCEPTIBILITY TO VANCOMYCIN ASSOCIATED WITH REDUCED SUSCEPTIBILITY TO DAPTOMYCIN IN S. AUREUS

No. (%) of IsolatesVancomycin Daptomycin

DaptomycinMIC, mcg/ml MIC < 1 mcg/ml MIC > 2

mcg/ml

< 2 812 (97) 30 (3)4 11 (20) 43 (80)8-16 1 (7) 15 (93)> 32* 5 (100) 0

(0)

* vanA mediated resistanceClin Infect Dis 2006;42:1652-3

CDC REPORT CASE.. 4 years old girl admitted with aplastic

anemia and fever. Started on cefotaxime

Week 1:Blood cultures + E.coli isolates R TO amp/ narrow spectrum

cephalosporines. Week2-4: persistent fever and bacterimia

? Other suspected organism..no, only EC Varieties of antimicrobial: genta, ticarcillin,

cefotaxime, ceftazidime, vanco, clinda.. Patient was not improved…micro lab

contacted..

TEM-1: LRSHV-1:LR-CAZSHV-8:HLR-CAZ

During a period of <2 months in the blood stream of 4 years old patient, an EC strain :•acquired a new beta lactamase gene that mediated R 3rd ceph(SHV-1)…

•mutated the gene to increase the level of ceph-R(SHV-8) …

•and down regulating its cell wall porins(OmpF) to increase R not only to cephalosporins but cephamycins as well….

SUMMARY

Development of antimicrobial resistance is directly related to improper antimicrobial choice, dose, and duration.

Understanding antimicrobial pharmacokinetics/dynamics and resistance mechanisms can help guide appropriate usage.

Knowledge of local susceptibility patterns is essential.

Paucity of new antimicrobial agents in pipeline

THANK YOU….