Carbapenamases Facts and Detecction

8/7/2019 Carbapenamases Facts and Detecction

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CARBAPENAM

ASESFacts, Controversies,DetectionDr.T.V.Rao MD


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Dr.T.V.Rao MD 2

ang ng an scape orNumbers of Approved Antibacterial

Agents

We have more resistant Microbes

Bars represent number of new antimicrobial agents approved by the FDA during the.p eri od l is te d

002468

1012141618

-1983 87 -1988 92 -1993 97 -1998 02 -2003 05 2008

.Infectious Diseases Society of America ,Bad Bugs No Drugs. ; .July 2004 Spellberg B et al Clin Infect Dis. ; : - ;2004 38 1279 1286.New antimicrobial agents Antimicrob Agents Chemother. ; :2006 50 1912


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Dr.T.V.Rao MD 3

Carbapenems x

PenicillinThe carbapenems arestructurally verysimilar to thepenicillins, but thesulphur atom in

position 1of thestructure has been

replaced with acarbon atom, andhence the name ofthe group, the

carbapenem


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Dr.T.V.Rao MD 4

What are carbapenems

Carbapenems are a class ofbeta-lactam antibiotics with abroad spectrum of

antibacterial activity. Theyhave a structure that rendersthem highly resistant to beta-

lactamases. Carbapenemantibiotics were originallydeveloped from thienamycin,

a naturally-derived product of


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Spectrum of activity

Broad spectrum activity GPC & GNB

Aerobic & Anaerobic bacteria

Active against MDR isolates

Active against ESBL +ve GNB

Active against Ps aeruginosa &Acinetobacter spp.

Not active against MRSA

Enterococcus spp.

Stenotrophomonas maltophilia


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Dr.T.V.Rao MD 6

Carbapenems inCommon Use

Imipenem Broad spectrum, covers Gram-positive,

Gram-negative (including ESBL-producing strains), Pseudomonas and

anaerobes Meropenem

Less seizure-inducing potential, canbe used to treat CNS infections

Ertapenem Lacks activity vs. Acinetobacter and

Pseudomonas Has limited activity against penicillin-

resistant pneumococci


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Dr.T.V.Rao MD 7

Carbapenems effective onseveral common isolates

Staph(notMRSA), Strep(highlyresistant),Neisseria,Haemophilus,Proteus,

Pseudomonas,Klebseilla,Bacteroides,anaerobes(excluding C.dif)


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Dr.T.V.Rao MD 8

Spectrum of Activity

Drug Strep spp. &MSSA

Entero-bacteriaeae

Non-fermentors

Anaerobes

Imipenem + + + +

Meropenem + + + +

Ertapenem + + Limitedactivity

+

Doripenem + + + +


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Dr.T.V.Rao MD 9

Carbapenems

Drug Route of Administration

FDA Status

Imipenem IV Cleared

Meropenem IV Cleared

Ertapenem IM, IV Cleared

Doripenem IV Application Submitted


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Dr.T.V.Rao MD 10

Enterobacteriaceae are realproblamatic microbes

The rapid anddisturbing spreadof:

ESBL extended-spectrum -lactamases

AmpC enzymes carbapenem

resistance metallo--

lactamases

0 1 0


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:0 1 0 Distribution of lactamases according to fu

Most Carbapenemases canHydrolyzeALLBeta lactam antibiotics


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Discovery ofCarbapenamases

In 1996, the first isolate of KPC-producing bacteria was discovered ina clinical specimen ofK pneumoniae

from a hospital in North Carolinainvolved in the Intensive CareAntimicrobial ResistanceEpidemiology (ICARE) surveillance

program. KPCs were infrequentlyisolated until 2001, when KPC-producing Enterobacteriaceae werereported in several extended

outbreaks in metropolitan hospitals ofNew York and New Jerse .


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Carbapenems used asimportant life saving option

Carbapenems are often used asantibiotics of last resort for treatinginfections due to multidrug-resistant

gram-negative bacilli, because theyare stable even in response toextended-spectrum and AmpC -lactamases. However, gram-

negative bacilli producing theacquired metallo--lactamases (MBLs)IMP and VIM have been increasinglyreported in Asia and Europe and

more recently, they have beendetected in Canada and the United


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Carbapenemases

The most versatile family of -lactamases

Two major groups based on the hydrolytic

mechanism at the active site Serine at the active site: class A and D

Zinc at the active site : class B

All carbapenemases hydrolyze penicillin's,extended spectrum cephalosporins, andcarbapenems


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Dr.T.V.Rao MD 16

Carbapenamases arespreading faster

A new class of bacterial enzymescapable of inactivatingCarbapenems, known as

Klebsiella pneumoniaeCarbapenamases (KPCs), hasrapidly spread in the United States

and continues to be extensivelyreported elsewhere in the world.KPCs are class A Carbapenamasesthat reside on transferableplasmids and can hydrolyze all


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Dr.T.V.Rao MD 17

Carbapenemases withinthe Enterobacteriaceae

KPC carbapenemase Difficult todetect using current MICbreakpoints.

Isolates that have an MIC of 2 g/mlto ertapenem or an MIC of 2-4 g/ml to meropenem or Imipenem.

Modified Hodge test isconfirmatory..PCR is gold standard.


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Dr.T.V.Rao MD 18

KPC(K. pneumoniaecarbapenemase)

KPCs are the mostprevalent of thisgroup of enzymes,

found mostly ontransferable

plasmidsinK.

pneumoniae

Substratehydrolysis

spectrum


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Dr.T.V.Rao MD 19

KPCs in

EnterobacteriaceaeSpecies CommentsKlebsiella spp. K.pneumoniae-cause of outbreaks

K.oxytoca-sporadic occurrence

Enterobacterspp. Sporadic occurrenceEscherichia coli

Salmonella spp.

Citrobacter freundii

Serratia spp.


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Pseudomonas aeruginosaCarbapenamases

KPC resistance hasbeen reported ininherently resistantorganisms such as

Pseudomonasfrom Trinidad, an

isolate of multidrug-resistantPseudomonasaeruginosa that

harboured a novel


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Enzyme Ambler

Class

Country Spectrum of activity Organisms

IMP (18)ImipenemJapan

B Japan All lactams Ps., Acinetobacter

VIM (12)Verona

B Italy Pan R, may be S toaztreonam

Ps. , Acinetobacte

SPMSao Paulo

B Brazil Pan R Ps

GIM German B Germany Pan R Ps.

SIMSouel

B SouthKorea

Pan R Acinetobacter, Ps.

NDMNew Delhi

B India, UK Pan R Kleb pneu, E.

coli

M e ta llo (lactamases Zn at)a ctiv e site


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Dr.T.V.Rao MD 25

Carbapenemase Class A

First identified 1982 in UK

Four major families

Chromosomally encoded Serratia marcescens enzyme (SME)

Not metalloenzyme carbapenemases (NMC)

Imipenem-hydrolyzing -lactamases (IMI)

Plasmid encoded

Klebsiella pneumoniae carabapenemases (KPC)

Guiana Extended-Spectrum (GES)


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Dr.T.V.Rao MD 26

Emerging CarbapenemResistance in Gram-Negative

Bacilli

Significantly limits treatment optionsfor life-threatening infections

No new drugs for gram-negative bacilli

Emerging resistance mechanisms,carbapenemases are mobile,

Detection of carbapenemases andimplementation of infection controlpractices are necessary to limit

spread

E b i B k i


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Dr.T.V.Rao MD 27

Enterobacteriaceae: Breakpointsrevised so need for other newer

drugs, may be carbapenms ?

AgentCLSI 2009 CLSI 2010

S I R S I R

Cefazolin 8 16 32 1 2 4

Cefotaxime 8 16-32 64 1 2 4

Ceftriaxone 8 16-32 64 1 2 4

Ceftazidime 8 16 32 4 8 16

Aztreonam 8 16 32 4 8 16

Cefipime 8 16 32 8 16 32


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Laboratory Detection: &al and Laboratory Standards Institute breakpoints 2009 201Revised Break Points 2010Agent MIC breakpoint (ug/ml) DD breakpoints (mm)

S MHT I R S MHT I R

IPM < 1< 1

2-4 82

>16>4

>16>23

NA 14-1520-22

4

>22>23

16-21 14-1520-22

1

> 22>23

19-21 16-1820-22


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Dr.T.V.Rao MD 29

Class ACarbapenemases

K. p neumoniae carbapenemase( - ) -KPC type possess carbapenemhydrolyzing enzymes most

. .common on East Coast of U S Enzymes are capable ofefficiently hydrolyzing

, ,penicillins Cephalosporins,aztreonam and carbapenems

and are inhibited byclavulanic acid andtazobactam

To date 4 KPC enzymes have

: - , - ,been identified KPC 1 KPC 2- - Cl A


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Dr.T.V.Rao MD 30

Class ACarbapenemases

K. p neumoniae carbapenemase( - ) -KPC type possess carbapenemhydrolyzing enzymes most

. .common on East Coast of U S Enzymes are capable ofefficiently hydrolyzing

, ,penicillins Cephalosporins,aztreonam and carbapenems

and are inhibited byclavulanic acid andtazobactam

To date 4 KPC enzymes have

: - , - ,been identified KPC 1 KPC 2- -


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Dr.T.V.Rao MD 31

Located on plasmids; conjugativeand nonconjugative

blaKPC is usually flanked bytransposon sequences

blaKPC reported on plasmids with:

Normal spectrum -lactamases Extended spectrum -lactamases Aminoglycoside resistance

KPC Enzymes


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Dr.T.V.Rao MD 32

When to Suspect a KPC-Producer

Enterobacteriaceae especiallyKlebsiella pneumoniae that areresistant to extended-spectrum

cephalosporins: MIC range for 151 KPC-producing

isolates Ceftazidime 32 to >64 g/ml

Ceftriaxone 64 g/ml

Cefotaxime 64 g/ml

Variable susceptibility to cefoxitin and

cefepime


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Modified Hodge Test

for CarbapenemaseDetection in

Enterobacteriaceae


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Laboratory Detection ofKPC-Producers

Problems: 1) Some isolates demonstrate low-level

carbapenem resistance

2) Some automated systems fail to

detect low-level resistance

Th M difi d H d


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The Modified HodgeTest

The Modified Hodge Test is aphenotypic confirmatory test forCarapnemase activity and is

indicated when there is a positivescreening test and resistance to oneor more agents in cephalosporin

subclass III (i.e., cefoperazone,cefotaxime, ceftazidime,ceftizoxime, and ceftriaxone) Beaware that imipenem disk tests

perform poorly as a screen for

Phenotypic Tests for


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Phenotypic Tests forCarbapenemase

Activity

Modified Hodge Test

100% sensitivity in detecting KPC; alsopositive when other carbapenemasesare present

100% specificity

. , , - .Procedure described by Lee et al CMI 7 88 102

.2001

Th M difi d H d


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The Modified HodgeTest (MHT)

The Modified Hodge Test (MHT)detects carbapenemase production

in isolates of Enterobacteriaceae Carbapenemase production is

detected by the MHT when the test

isolate produces the enzyme andallows growth of a carbapenemsusceptible strain (E.coli ATCC25922) towards a carbapenem disk

Step 1 and 2 of


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Step 1 and 2 of

MHT Prepare a 0.5McFarlanddilution of the

E.coli ATCC25922 in 5 ml ofbroth or saline.

Dilute 1:10 byadding 0.5 ml ofthe 0.5McFarland to 4.5

ml of MHB or


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Step 3 and 4 of MHT

Streak a lawn ofthe 1:10 dilutionofE.coli ATCC25922 to a

Mueller Hintonagar plate andallow to dry 35minutes.

Place a 10 gmeropenem orertapenem

susceptibility disk


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Protocols in ModifiedHodge Test


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Step 5 and 6 of MHT

In a straight line, streak testorganism from the edge of thedisk to the edge of the plate.Up to four organisms can betested on the same plate withone drug.

Incubate overnight at 35C 2OCin ambient air for 1624 hours


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Detection

Anderson KF et al. Evaluation of methods to identify KPC inenterobacteriaceae. JCM 2007; 45: 2723 2725.

Modified Hodge Test (MHT) Carbapenem Inactivation Assay

Carbapenem Disk

SusceptibleE. coli

Test Isolate

M difi d H d T


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Modified Hodge Test

Lawn ofE. coliATCC 259221:10 dilution of a0.5 McFarland suspension

Imipenem disk

Test isolates

. , , -Described by Lee et al CMI 7 88. .102 2001

serva on or


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serva on orCarbapenamases

detection by HMT After 1624 hours

of incubation,

examine theplate for a cloverleaf-typeindentation atthe intersection

of the testorganism and theE. coli 25922,within the zone

of inhibition of

Q li l i i


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Quality control strains inModified Hodge test

Perform quality control of theCarbapenems disks according to

CLSI guidelines.

Perform quality control with each run.

MHT Positive Klebsiellapneumoniae ATCC BAA-1705

MHT Negative Klebsiellapneumoniae ATCC BAA-1706

y es ng w


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y es ng wErtapenem or

Meropenem The procedure described by

Landman et al. describes using a

10-g imipenem disk for step 1.However, there are species ofEnterobacteriaceae which haveintrinsic mechanisms of resistance

to imipenem other than acarbapenemase (See CLSIdocument M100, Appendix G).Therefore, ertapenem ormeropenem may provide more

specific selection for acquired

What Labs Should Do


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What Labs Should DoNow

Look for isolates ofEnterobacteriaceae (especially K.pneumoniae), with carbapenem MIC

2 g/ml or nonsusceptible toErtapenem by disk diffusion

Consider confirmation by ModifiedHodge Test

Alert clinician and infection controlpractitioner to possibility of mobilecarbapenemase in isolate

Newer


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NewerCarbapenemases

As of June 2010, there werethree reported cases ofEnterobacteriaceae isolatesbearing this newly describedresistance mechanism in theUS, the CDC stated that "Allthree U.S. isolates werefrom patients who receivedrecent medical care in

"


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K. pneumoniae containing NDM-1was first discovered in 2008. By2009, a study in Mumbai revealed

24 carbapenem-resistantEnterobacteriaceae, 22 of whichwere NDM-1 producers. Of these 22organisms, 10 were klebsiella

species, 9 were Escherichia coli, 2were enterobacter species, and 1was Morganella morganii illustrating the ability of the

plasmid to spread rapidly among

NDM-1

CDC t th


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CDC reports the newgenetic mechanisms

The isolate, Klebseilla pneumoniae05-506, was shown to possess ametallo-beta-lactamase (MBL)

but was negative for previouslyknown MBL genes. Gene librariesand amplification of class 1integrons revealed three

resistance-conferring regions; thefirst contained bla(CMY-4) flanked by ISEcP1and blc. The second region of 4.8 kbcontained a complex class 1 integron

with the gene cassettes arr-2, a new

N D lhi t ll b t


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NDM-1, which stands for New Delhimetallo-beta-lactamase 1 and actuallyrefers not to a single bacterial speciesbut to a transmissible geneticelement encoding multiple resistancegenes that was initially isolated froma strain of Klebsiella obtained from apatient who acquired the organism inNew Delhi, India

Subsequently, organisms in theEnterobacteriaceae family containing thisgenetic element (or variants thereof) have

been found widely throughout India,

New Delhi metallo-beta-lactamase 1


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NDM-1 NDM-1 symptoms are reported to

be associated with the bacteria itattaches to. The currently knownbacteria's hosting this gene are E.Coliand Klebsiella pneumoniae. Themajority of the patients treated todate who are positive for NDM-1 werethose with urinary tract infections,bacteraemia, or pneumonia NDM-1 isthe gene responsible for the newestsuperbug. NDM-1 genes can live

inside different bacteria and is

w


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wDelhi creates

Controversy The gene was named after New Delhi, thecapital city of India, as it was firstdescribed by Yong et al. in 2009 in aSwedish national who fell ill with an

antibiotic-resistant bacterial infection thathe acquired in India . The infection wasunsuccessfully treated in a New Delhihospital and after the patient's

repatriation to Sweden, a carbapenem-resistant Klebsiella pneumoniae strainbearing the novel gene was identified. Theauthors concluded that the new resistancemechanism "clearly arose in India, but

there are few data arising from India to


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CDC reportsThree Enterobacteriaceae isolates

carrying a newly described resistancemechanism, the New Delhi metallo-

beta-lactamase (NDM-1) , wereidentified from three U.S. states atthe CDC antimicrobial susceptibility

laboratory. This is the first report ofNDM-1 in the United States, and thefirst report of metallo-beta-lactamasecarriage among Enterobacteriaceae

in the United States

Blame on India Is it


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Blame on India Is itjustified ?

As of June 2010, there werethree reported cases of

Enterobacteriaceae isolatesbearing this newly describedresistance mechanism in the

US, the CDC stated that "Allthree U.S. isolates werefrom patients who received

recent medical care in

CDC reports the new


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CDCreports the newgenetic mechanisms

The isolate, Klebseilla pneumoniae05-506, was shown to possess ametallo-beta-lactamase (MBL) but

was negative for previously knownMBL genes. Gene libraries andamplification of class 1 integronsrevealed three resistance-

conferring regions; the first containedbla(CMY-4) flanked by ISEcP1 and blc. Thesecond region of 4.8 kb contained acomplex class 1 integron with the gene

cassettes arr-2, a new erythromycin

Molecular configuration


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Molecular configurationof NDM-1

NDM-1 also has an additional insertbetween positions 162 and 166 notpresent in other MBLs. NDM-1 has a

molecular mass of 28 kDa, ismonomeric, and can hydrolyze allbeta-lactams except aztreonam.

Compared to VIM-2, NDM-1 displaystighter binding to mostCephalosporins.

NDM ti di diff f th


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NDM genetic coding differs from otherrecent isolates

Compared to VIM-2, NDM-1 displaystighter binding to mostcephalosporins, in particular,cefuroxime, cefotaxime, and

cephalothin (cefalotin), and also tothe penicillins. NDM-1 does not bindto the carbapenems as tightly as IMP-1 or VIM-2 and turns over thecarbapenems at a rate similar to thatof VIM-2. In addition to K. pneumoniae05-506, bla(NDM-1) was found on a140-kb plasmid in an Escherichia colistrain isolated from the patient's

feces, inferring the possibility of in


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CLSI guidelines for assessingthe antibiograms pattern

All patients colonized or infected withCRE or carbapenemase-producingEnterobacteriaceae should be placedon contact precautions. Acute carefacilities should establish a protocol,in conjunction with CLSI guidelines, todetect nonsusceptibility andcarbapenemase production inEnterobacteriaceae, particularlyKlebseilla spp. and Escherichia coli,and immediately alert epidemiologyand infection control staff members ifidentified

Ph t i d t ti ith H d


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Phenotypic detection with Hodgetest a Minimal requirement

Carbapenem resistanceand carbapenemaseproduction conferredby blaNDM-1 is

detected reliably withphenotypic testingmethods currentlyrecommended by the

Clinical andLaboratory StandardsInstitute , includingdisk diffusion testingand the modified

Hodge test


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CHROMagarESBL & KPC

Why is CRE a public health


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Why is CRE a public healthemergency ?

Significantly limits treatment optionsfor life threatening infections

No new drug for GNB in the pipeline

Resistant mechanism easilytransferable as it in now on atransposon

Rapid Detection & effective infectioncontrol measures essential tocontrol spread


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Testing Other Drugs

Polymyxin B or Colistin

Could test either, but colistin usedclinically

Disk diffusion test does not work dont use!

Etest works well, but not FDAcleared

Broth micro dilution reference labs

Breakpoints - none

MIC 2 g/ml, normal MIC range

MIC 4 g/ml indicates increased

Laboratories should


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Laboratories shouldcreate protocols for

detection of CRE The exact procedure for confirmation of

CRE or carbapenemase-production

should be laboratory-specific andchosen based upon laboratoryworkflow and the types of isolatescausing clinical infections in thepatient population served. It may behelpful to refer to the CLSI guidelinesfor identification of carbapenemase

production in isolates that test

u oma on as m e


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u oma on as m euse in Carbapenamases

detection Automated testingalone will notdetect all of theresistancepatterns that

occur via beta-lactamases andcarbapenemases.Failure to detectorganisms with

these enzymescan result inerroneous reportsthat wouldindicate an isolate

is susceptible to-

Become a Member of Alliance for


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Dr.T.V.Rao MD 66

the Prudent Use of Antibiotics(APUA) www.apua.org

An internationalorganization

dedicated tocurbingantibiotic

resistanceChapters exist

currently in

several Asian

an as ng an


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an as ng anReduce the Spread of

Microbes


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Created by Dr.T.V.Rao MD for e learning resources for theMedical Professionals in the

Developing Worldemail.

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