1

Resistance to beta-lactams in Enterobacteriaceae:

mechanisms and detection

Y. Glupczynski

National Reference Center for Antimicrobial Resistance of

Gram-negative bacteria, CHU Mont-Godinne-UCL, 5530 Yvoir

Classification

Extended-Spectrum β-Lactamases

(ESBLs)

• OXA

Class DClass A

• SHV

• TEM

mutations

Ser70

What is an ESBL?

Serine ß-lactamases Metallo-enzymes

Group 1 Group 2dGroup 2 Group 3

AmpC TEM/SHVCTX-M, Others

OXA IMP,VIM, GIM,SPM, SIM

CephsInhib-R

Pens CephsInhib-S

Pens (Oxa++)Inhib-R/S

CarbapenemsInhib-R

K Bush, curr. Opin. Invest. Drugs 3-1284 (2002)

ESBLs

Plasmid encoded

R to all ß-lactams, except temocillin, (cephamycins),

carbapenems

inhibited by clavulanic acid

Evolution of extended-spectrumβ-lactamases (ESBLs)

Activity vsoxyimino cephs(3rd Gen Cephs)

TEM-11964

TEM-21970

Gln39→→→→Lys

TEM-31987

Gln39→→→→Lys Glu104→→→→Lys Gly238→→→→Ser

First ESBL

Epidemiology of ESBLs

Pre –2000

Mostly Klebsiella spp. with TEM/SHV

Nosocomial, often ICU / specialist units

1998: c. 25% of Klebs from European ICUs ESBL+

Few epidemic strains - e.g K. pneumoniae K25 SHV-4 in France & Belgium

Occasional local outbreaks in Belgian hospitals(Brussels, Gent, Leuven)

c. 5-15% of Klebsiella spp.; 40-60% E. aerogenes;

<2% E. coli

Plasmid encoded

1983 SHV-type (>130)

1985 TEM-type (> 180)

1989 CTX-M-type (> 100)

1988 SFO-1 Serratia FOnticola

1991 TLA-1 TLAhuicas (indian tribe)

1991 PER (7) Pseudomonas Extended Resistance

1996 VEB (7) Vietnam Extended-spectrum ß-lactamase

1996 BES-1 Brazilian Extended-Spectrum ß-lactamase

1998 GES (16) Guyana Extended-Spectrum ß-lactamases

2005 BEL (2) Belgium Extended-spectrum ß-Lactamase

2005 TLA-2 ???? (Plasmid, waste water)

1998 KPC (10) Klebsiella pneumoniae Carbapenemase

1991 OXA-ESBL (18) (OXA-2, -10, -13 derivatives, OXA-18, OXA-45)

ß-lactamases (>600) > 50% ESBLs

“The oldies”

“ESBLs of growing importance”

“infrequent

ESBLs”

“peculiar

ESBLs”

� http://www.lahey.org/studies/webt.asp

� Naas et al., 2008, CMI, Suppl 1:42-52)

2

K. georgiana- related

K. ascorbata- related

CTX-M ββββ-lactamases

- Cefotaximases rather than ceftazidimases

- Predominant ESBLs in Argentina since 1990

- Disseminating worldwide since 2000’s

>100 types, 5 clusters: evolved via escape

of chromosomal β-lactamase genes from Kluyvera spp.

ESBLs in the 21st Century

Major shift from TEM/SHV to CTX-M

Often in E. coli; (variable degree of clonality)

Often in community patients with no/little HC contact

Deaths; high attributable mortality

CTX-M Producers often multi-resistant (quinolones, cotrimox, AGs);

-> major driver of carbapenem use

Epidemiology of CTX-M β-Lactamases Transmission

bla genes mobilized from chromosomes to plasmids:

• transposases, insertion sequences, integrases involved

• blaCTX-M mobilizes 10x more frequently than blaSHV & blaTEM

Horizontal & vertical transmission of plasmids:

• Horizontal transmission: plasmid-mediated conjugation

• Vertical transmission: clonal transmission by normal cell division

ESBL transmission

Diversity of Mobiles Genetic Elements (MGE)

carrying ESBL genes

Smet A et al. FEMS Microbiol Rev 2009

Transposon

blaTEM-144

IR IR

blaSHV-5 IS26IS26

Specific IS (ISEcp1), Complex integrons (ISCR1)

Phage related elements

blaCTX-M-15ISEcp1/tnpA

blaCTX-M-14ISCR1

blaCTX-M-10

blaTEM-52

Tn3

Tn2

Insertion sequence

TEM

SHV

CTX-M

ESBL transmissionESBL transmission routes

3

Classification

Extended-Spectrum β-Lactamases

(ESBLs)

• OXA

Class DClass A

• SHV

• TEM

mutations

Ser70

Carbapenemases

Rising threat of multi-drug antibiotic resistance

Back to the pre-antibiotic era

PLASMID-ENCODED CLASS A

KPC ENZYMES: KLEBSIELLA PNEUMONIAE CARBAPENEMASE

Located on large plasmids;

• conjugative and nonconjugative

blaKPC is usually identified within a Tn3-type transposon (Tn4001).

blaKPC reported on plasmids with:

• Normal spectrum b-lactamases

• Extended spectrum b-lactamases

• Aminoglycoside resistance

GENETIC APPARATUS OF TRANSFERABLE MBLs

Plasmids, integrons and transposons

carbapenemase-produing Enterobacteriaceae isolates

Escherichia coli Klebsiella pneumoniae

Increased mortality with Infections caused by KPC producers

• A high invasive-device score was a predictor of IRE isolation (P 0.02).

• The mortality in the IRE group was 33%, compared to 9% among controls (P 0.043)

• Delay in effective therapy did not affect the in-hospital mortality but was found to increase the length of hospitalization among survivors (34+/-3.2 days versus 26.5 +/-5.4 days; P 00.2)

Why are carbas in the top chart of ß-lactamases?

•Case patients were more likely than control patients to �die during hospitalization (48% vs 20%; P < .001) �die from infection (38% vs 12%; P < .001).

• The timely administration of antibiotics with in vitro activity against CR-KP was not associated with patient survival.

4

KPC: Klebsiella Pneumoniae Carbapenemase

Source: Patrice Nordmann, Thierry Naas, and Laurent Poirel . Global Spread of Carbapenemase producing

Enterobacteriaceae. Emerging Infectious Diseases , October 2011: Vol. 17, No. 10, pp. 1791-1798.

Mortality rate:

> 50%

VIM: Verona Integron–encoded Metallo-β-lactamase

Source: Patrice Nordmann, Thierry Naas, and Laurent Poirel . Global Spread of Carbapenemase producing

Enterobacteriaceae. Emerging Infectious Diseases , October 2011: Vol. 17, No. 10, pp. 1791-1798.

Mortality rate:

18-62%

NDM: New-Delhi Metallo-β-lactamase

Source: Patrice Nordmann, Thierry Naas, and Laurent Poirel . Global Spread of Carbapenemase producing

Enterobacteriaceae. Emerging Infectious Diseases , October 2011: Vol. 17, No. 10, pp. 1791-1798.

Balkan countries, Middle east

Indian subcontinent

North Africa

OXA-48 : OXAcillinase

Source: Patrice Nordmann, Thierry Naas, and Laurent Poirel . Global Spread of Carbapenemase producing

Enterobacteriaceae. Emerging Infectious Diseases , October 2011: Vol. 17, No. 10, pp. 1791-1798.

Evolution of the distribution of resistance mechanisms of carbapenemase-

producing Enterobacteriaceae isolates, National Reference Centre, Belgium,

January 2007–October 2011 (n=80)

0

5

10

15

20

25

30

35

40

45

50

2007 2008 2009 2010 2011 oct

Year

Nu

mb

er

of

CP

E i

so

late

s O XA -48

K P C -2

NDM-1

V IM-2

V IM-1

x3

x3

x3

The rule of 3… as rule of thumb

With courtesy from T-D. Huang

Geographical origin (by country of cross-border transfer) and type

of carbapenemase-encoding genes detected in patients carrying

carbapenemase-producing Enterobacteriaceae,

Belgium, January 2007–October 2011 (n=73)

Patients Carbapenemase

Country /

Region VIM-1 VIM-2 NDM-1 KPC-2 OXA-48

VIM-1

and

KPC-2 Total

No travel

abroad 11 1 4 35 51

Greece 3 6 1 10

Italy 1 3 4

Morocco 3 3

Iraq 1 1

Kosovo 1 1

Montenegro 1 1

England 1 1

Pakistan 1 1

Total 16 1 3 13 39 1 73

5

Types of CPE isolates recovered in different

hospitals (n=80)

0

5

10

15

20

25

A B C D E F G H I J K L M N O P Q R S T U V W X

Hospital

Nu

mb

er

of

iso

late

s

VIM-1 VIM-2 NDM-1 KPC-2 OXA-48

With courtesy from T-D. Huang

Heterogeneous expression of MBLs

in Enterobacteriaceae

Enterobacteriaceae MIC (µg/ml)

species MBL Imipenem Meropenem

S. marcescens IMP-type

VIM-type

32- >128

64

128- >128

64

K. pneumoniae IMP-type

VIM-type

0.25- >128

2

0.25- >64

0.5

Enterobacter sp. IMP-type 0.25-8 0.25-8

Citrobacter sp. VIM-type

E.coli, S. flexneri 1-8 0.25-4

E. coli,

K. pneumoniae

NDM-1 >32 >32

C. freundii KHM-1 2 4

Adapted from P. Nordmann; Arch Ped 2010; 17: S156-S162

Heterogeneous expression of KPC

carbapenemase in EnterobacteriaceaeOXA-48 carbapenemase (Class D)

Plasmid-mediated resistance Mostly in Klebsiella spp. (E. coli)

Turkey, India, North AfricaUK, France, Belgium (since 2010)

Variable level of resistance to carbapenems

(meropenem MICs <0.5->128)

Susceptible to 3-4 gen cephs when not

Associated with other mechanisms

Resistance to penicillins (temocillin

incl.); no effect of BL inhibitors

Difficult to detect (low-level R,

algorithms of Expert systems not adapted)

Y. Glupczynski, personal data

29

Meropenem MIC: KPC- and MBL-isolates

0

2

4

6

8

10

12

14

16

0,032 0,064 0,125 0,25 0,5 1 2 4 8 16 >=32

MIC

No

iso

late

s

MP KPC

MP MBL

WT

K. pneumoniae and meropenem

CLSI 2010 4 / 8

CLSI 2011 1 / 4EUCAST 2 / 8

PHENOTYPIC CONFIRMATION

1. Modified Hodge Test2. Meropenem-APBA comb. Disks

3. Meropenem-DPA comb. Disks4. Imipenem-EDTA comb. Disks

Guidelines for phenotypic screening and confirmation of carbapenemases in

Enterobacteriaceae

Carbapenemase Screening:

E. coli, Klebsiella spp., Salmonellaspp., Enterobacter spp, Citrobacter spp.

Expert system positive ORMIC meropenem > 0.5 mg/L; Mero (10 µg)Disk zone < 23 mm) OR MIC imipenem > 2 mg/L;Imip (10 µg) disk zone < 21 mm)

No carbapenemase

Elevated carbapenem MIC confirmed with meropenem or imipenem Etest ?

GENOTYPIC CONFIRMATION

Cohen-Stuart et al., Int J Antimicrob Agents 2010; 36:205-10

Carbapenemase Screening:

Proteus spp., M. morganii, Providenciaspp., Serratia spp.

Expert system positive ORMIC meropenem > 0.5 mg/L; Mero (10 µg) disk zone < 23 mm)

6

K. pneumoniae KPC-positive (class A

carbapenemase)

IMI+DPA IMI IMI+EDTAMERO + APBA

MERO+DPA MERO MERO+CLMERO+EDTA

MEROMERO +DPA MERO +CL

MERO +BOR

ERTAIMI IMI + EDTA

KPC-2

With courtesy from TD Huang

DDST for detection of MBLs

IMI IMI + EDTA

EDTA (420 µg)

1.

2.

NDM-1

Detection of asymptomatic carriage of carbapenemase in the gut flora

From rectal swabs (stools)

Chromogenic selective culture media- Chrom ID BLSE (cefpodoxime)

- CHROMagar KPC (Carbapenem)- Brilliance CRE (Oxoid) (Carbapenem)

- ChromID CARBA (bioMérieux) (Carbapenem)

- MAC+ IMI (1 mg/L)- MAC + 10-µg disks (ERTA/MERO/IMP)

Limit of sensitivity of detection (102 to 103)

(Chrom ID BLSE > CHROMagar KPC)

Enrichment broth culture not superior to direct plating

CHROMagar KPC(carbapenemase producers)

Gold standard: molecular biology +++

Multiplex PCR; IMP, VIM, KPC, OXA-48, NDM….

NDM-1, KPC, AmpC and ESBL Check-MDR CT101*

NDM-1 KPC, OXA-48, VIM, IMP and ESBL Check-MDR CT102*

Molecular confirmatory tests

(Naas et al. AAC, 2010; JCM, 2011)

Spectrophotometric assays for Carbapenem-hydrolysis +++

Rapid Molecular detection tools

ß-lactamases (>600) > 50% ESBLs

SHV-type (> 150), TEM-type (> 190), CTX-M-type (> 100)

TLA-1, PER (7) , VEB (7), BES-1, GES (17), BEL (3), TLA-2, KPC (10), OXA-ESBL

PCR and sequencing•The gold standard• Can detect all variants• Easy to perform but labor intensive and costly

� PCR, Multiplex-PCR, Real-time PCR (Sybr green, Taqman, Hybridization probes), Real time

PCR coupled with pyrosequencing, PCR-ESI (Maldi)….⇒ Only seeked genes can be found ⇒ Only detection of DNA (not expressed genes)

⇒ Requires isolation of the strain for identification and susceptibility testing⇒ Most of the presently used assays are confirmative tests (on cultures)

detection-ligation

PCR

Hybridisation

detection

7

Array: summary

DNA

extraction

1 h

Identification

Amplification

3 h

Detection

2 h

Results

on line

Hands on time:- 3 samples: 1 h

-15 samples: 1 ½ h- 72 samples: 2 ½ h

72 samples/day, cost effective run: minimum 3 samples Patient transferred from Montenegro –

Colonization (resp.) – Clinical outcome: curedPatient transferred from Pakistan –Wound infection – Clinical outcome: deceased

Example: 2 patients in western Europe with pan-drug resistant Enterobacteriaceae isolates

Escherichia coli Klebsiella pneumoniae

Case: 2 Patients in western Europe

K.pneumoniae from

wound infection

(NDM-1; SHV-12; CTX-M-1 gr)

E.coli from faeces

(NDM-1; CMY-2 like

like; CTX-M-1 gr)

NDM-1

CTX-M- group1

SHV-12

CMY-2 like

Combination of resistance mechanisms detected in single experiment

Distinction between TEM/SHV ESBLs or non ESBLsCourtesy from W. de Levita

Result ESBL - KPC Array:

Naas et al. AAC 2010

Evaluate novel molecular assays

Molecular test based on micro-array (results in < 7 h)

Identification of ESBLs

• of TEM and SHV type

• non-ESBL TEM and SHV type

• CTX-M type

Identification of KPC

and KPC, OXA-48, NDM, VIM, IMP

and KPC, NDM, CMY-1, CMY-2, ACT, ACC, DHA, FOX

and KPC, OXA48, NDM, VIM, IMP, CMY-1 /-2, ACT, ACC, DHA, FOX

Cohen S, JAC, 2010

Naas, AAC, 2010Endimiani, JCM, 2010

Naas, JCM, 2011

Bogaerts, AAC

Multiplex end-point PCR assays

Genotypic detection of carbapenemase-encoding genes and other important β-lactamases (ESBLs, AmpC, OXA…)

Multiple protocols (3-7 sets, 11-25 target genes)

Design of specific group of primers

• Definition of different reaction mixtures

• Optimization of PCR conditions (one single amplification protocol)

• Distinct amplicon sizes for the different genes

Rapid (<4 h), reliable (specific)

Screening of clinical isolates, epidemiological surveys

Customizable to local needs (endemic/non endemic settings)

Lower costs (Vs multiple PCR assays)

Dalenne et al., JAC 2010Poirel et al., Diag. Microb. Infect Dis 2011Voets et al. IJAA 2011

8

Diag Microbiol Infect Dis 2011; 70: 119-123

Rapid (<4 h), reliable (specific)Screening of clinical isolates, epidemiological surveysCustomizable to local needs

(endemic/non endemic settings)Lower costs (Vs multiple PCR assays

Real-time Multiplex PCRDesign of specific primers and fluorescent probes (TaqMan)

Rapid (<2 h), one amplification reaction in single tube

Less prone to contamination (closed system, no tube opening)

Different ways for identification of amplicons

− Real-time probe-based PCR

− Amplicon melt-curve analysis (intercalating dye – LCGreen)

− Capillary electrophoresis of amplicons (QIAxcel –QIAGEN)

− DNA sequencing of amplicons (confirmation, identification of specific alleles –e.g: GES 170 Gly-Ser with carbapenemase activity)

Would allow more rapid guidance of antibiotic treatment and infection control measures

Swayne et al., IJAA 2011

Real time pentaplex PCR

Melt-curve analysis

of amplicons - LCGreenCapillary electrophoresis

of amplicons

Cycles

A

Fluor

esc

enc

e

106 105 104 103 102 101 100

No NDM-bacteria

B

Fluor

esc

enc

e

Cycles

106 105 104 103

102

101

No spiked NDM-bacteria

NDM-1-qPCRA: Serially diluted Kp NDM-1 in 108 E. coli WT

B: Serially diluted Kp NDM-1 in stool

Number of Kp NDM-1 in PCR tube

Number of Kp NDM-1 / 100 mg of stool

T Naas*, A Ergani, A Carrer, and P Nordmann, AAC 2011

Isolate

#

Bacteria MIC

(µg/ml)

Lowest limit of

detection in water

(CFU/ml)

Lowest limit of detection in

stool (CFU/ml of stool)a

Lowest detection limit of NDM-1

qPCR

IMPa ChromID

ESBL

CHROMag

ar KPC

ChromID

ESBL

CHROMagar

KPC

(CFU/ml of

stoola)

(DNA copies/ml

of stoolb)

23 K. pneumoniae A >32 1 x 101 1 x 10

1 2 x 10

1 1 x 10

2 10

1 1 x 10

2

24 K. pneumoniae B >32 2 x 101 1 x 10

1 2 x 10

1 5 x 10

1 2 x 10

1 2x 10

2

25 K. pneumoniae D >32 3 x 101 2 x 10

1 2 x 10

1 2 x 10

1 10

1 4 x 10

2

26 K. pneumoniae F >32 1 x 101 2 x 10

1 1 x 10

1 2 x 10

1 2 x 10

1 2 x 10

2

27 K. pneumoniae G 2 2 x 101 3 x 10

2 1 x 10

2 4 x 10

3 10

1 2 x 10

2

28 K. pneumoniae I >32 1 x 101 3 x 10

1 2 x 10

1 6 x 10

1 10

1 6 x 10

2

29 C. freundii A >32 1 x 101 1 x 10

1 1 x 10

1 1 x 10

1 3 x 10

1 6 x 10

2

30 E. coli A 6 2 x 101 4 x 10

1 2 x 10

1 3 x 10

2 10

1 2 x 10

2

31 E. coli B 3 1 x 101 2 x 10

3 1 x 10

1 3 x 10

3 10

1 2 x 10

2

32 E. cloacae A 8 4 x 101 3 x 10

2 2 x 10

1 8 x 10

1 3 x 10

1 10

3

Real-Time PCR for Detection of

NDM-1-carbapenemase genes from Stools

T Naas*, A Ergani, A Carrer, and P Nordmann (submitted AAC)

Real-Time PCR for Detection of

NDM-1-carbapenemase genes from Stools

Highly specific and sentitive qPCR (limit of detection +/-101 bacteria /

100 mg of stool=> use of enrichment culture (increased detection?)

Useful tool for outbreak management (rapid cohorting of patients, infection control measure evaluation, ….)

At least as sensitive as culture on stool samples, but 4h turn around time vs 48h-72h for culture

Needs to be evaluated on rectal swabs &/ in outbreak situationExtraction can be automated (less hands on time)

9

Multiplex PCR-ELISA

Direct Screening for MRSA / ESBL / VRE from 1 swab!Enrichment by culture is not necessary!Economical test system for 1 to 94 samples per run Simple test performance; Automation possible hyplex® TAT: 2-4 hoursReliable, secure, fast

1. DNA extraction2. Multiplex PCR3. Reverse hybridization of PCR products

to specific capture oligonucleotide probes

4. Detection of hybridization complex by peroxidaseconjugated antibody

Results in less than 2 hours!

hyplex® (Multiplex-PCR-ELISA)

Multiplex PCR-ELISA Evaluation of hyplex® MBL ID Multiplex PCR-ELISA(AmplexDiagnostics)

Avlami et al., J Microbiol Methods 2010; 83: 185-187

Clinical specimens (n) MBL-producers (n) Hyplex (+)

TP TN FP FN Sens (%)

Spec (%)

PPV (%)

NPV (%)

Blood (n=90) P. aeruginosa (8)K. pneumoniae (9)P. stuartii (1)E. aerogenes (1)

19 19 71 0 0 100 100 100 100

Other specimens (n=236)- Urine (60)- Bronchial/sputum (n=85)

P. aeruginosa (2)K. pneumoniae (3)P.aeruginosa (26)A. baumannii (6)K. pneumoniae (6)P. stuartii (2)

58742

53540

1775343

522

100

98 97 91 99

- Wound/Pus (n=91) P. aeruginosa (6)K. pneumoniae (2)E. aerogenes (1)

9 8 81 1 1

Total (n=326) 77 72 248 5 1 98.6 98 93.5 99.5

Identification of MBL (VIM/IMP) genes directly from clinical specimens326 samples, 3 hospitals (Athens)

73 VIM + (22.4%); no IMP found

Timely, reliable detection of MBL-producers

Need for multicentric studies (lower prevalence settings, IMP-positive organisms)

Conclusions

The epidemiology of carbapenemase (sources of acquisition, risk

factors, real prevalence/incidence) is poorly known and most

probably largely underestimated because of difficulties in their detection. It is obvious that carbapenemase–producing organisms

are distributed worldwide and their prevalence is increasing

•This rapid spread of carbapenemases will be a major heathcare issue for the next decades

•The « fight » against MDR carbapenemase-producing bugs, will require:

•Fast detection and identification of the reservoirs

•Reduced (appropriate) consumption of antimicrobials

•Strict adherence to standard hygiene practice

Acknowledgments

CHU Mont Godinne (UCL)

National Reference Laboratory

- Daniel Huang

- Alexia Verroken

- Pierre Bogaerts

- Caroline Bauraing

- Catherine Berhin

- Roberta Rezende de Castro

- Warda Boucharhouf

- Amélie Guisset

- Farid El Garch

Hôpital ULB-Erasme

Associated Ref. laboratory

- Ariane Deplano

- Sandrine Roisin

- Claire Nonhoff

- Ricardo De Mendonca

- Marie Hallin

- Olivier Denis

Institute of Public Health

- Bea Jans

- Boudewijn Catry

- Sophie Quoilin

MONT-

GODINNE