PCR based detection of Carbapenamases:

Different primers and their utility

Philma Glora. M&

Bhoj R SinghDivision of Epidemiology

Indian veterinary Research Institute, Izatnagar- 243 122, India

CarbapenamsWide spectrum, β lactam class of antibioticsDrug of last resort available as: imipenam, meropenam,

doripenam and ertapenam. Resistance to these drugs is emerging and may be

mediated through:Efflux mechanism.Loss of porins.Change/ alterations in PBPs.βlactamases - common (carbapenamases): These are

classified in to 4 Classes.

Class A CarbapenemasesSerine residue at active site of enzymePlasmid/chromosomeGenerally inhibited by clavulanic acidExamples

Chromosomally encoded Plasmid encodedSME (Serratia marcescens enzyme) GES (Guiana extended spectrum)

NMC (Non-metalloenzyme carbapenamase) KPC (Klebsiella pneumoniae carbapenamase)

IMI (Imipenam hydrolyzing β-lactamase)

Class B CarbapenemasesZn at enzyme active siteInhibited by EDTALocated in integrons/plasmidExamplesVIM (Verona integron encoded β-lactamase) SMP (Sao Paulo metallo β-lactamase)NDM1 (New Delhi metallo β-lactamase)IMP (Active on Imipenam)

Class C CarbapenemasesLow potential of carbapenam resistance when combined

with efflux mechanism over expressionpYMG-1

Class D CarbapenemasesOxacillin hydrolysisSerine at active site of enzymePlasmid encodedCommon in Acinetobacter spp.Poorly inhibited by EDTA and clavulanic acidExamples-OX-23, OX-48

Class

Producer Organisms

Hydrolysis profile of the enzymes

Penicillin

Early cephalosporins

Extended Spectrumcephalosporins

Aztreonam

Carbapenams

A Member of Enterobacteriaceae, Pseudomonas, Klebsiella

+ + + + (except GES)

+

B Pseudomonas, members of Enterobacteriaceae, Acinetobacter

+ + + - +

D Acinetobacter

+ + ± - ±

Detection of Carbapenemases withPCRNucleic acid-based detection systems: Rapid

and sensitive to detect the presence of resistance genes.

Play a critical role in the elucidation of resistance mechanisms & molecular epidemiology.

PCR involves cycles of heating the sample for denaturing, annealing of the primers, and elongation of the primers by a thermostable DNA polymerase.

Enzyme Family/ Gene

Primer Primer Sequence (5’-3’) Fragment Size (bp)

Entire coding region

Reference

Class A Carbapenameses

NMC NMC1NMC4

GCATTGATATACCTTTAGCAGAGACGGTGATAAAATCACACTGAGCATA

2,158 Yes Radice et al., 2004

SME IRS-5IRS-6

AGATAGTAAATTTTATAGCTCTAACGCTAATAG

1,138 Yes Queenan et al., 2000

IMI IMI-AIMI-B

ATAGCCATCCTTGTTTAGCTCTCTGCGATTACTTTATCCTC

818 No Aubron et al., 2004

KPC KPC forwardKPC reverse

ATGTCACTGTATCGCCGTCTTTTTCAGAGCCTTACTGCCC

893 Yes Bradford et al., 2004

GES GES-CGES-D

GTTTTGCAATGTGCTCAACGTGCCATAGCAATAGGCGTAG

371 No Weldhagen et al., 2004

Enzyme Family/ Gene

Primer Primer Sequence (5’-3’) Fragment Size (bp)

Entire coding region

Reference

Class B MetalloenzymesIMP Forward

ReverseCTACCGCAGCAGAGTCTTTGAACCAGTTTTGCCTTACCAT

587 Senda et al., 1996

IMP-1 ForwardReverse

TGAGCAAGTTATCTGTATTCTTAGTTGCTTGGTTTTGATG

740 Yes Yan et al., 2001

IMP-2 ForwardReverse

GGCAGTCGCCCTAAAACAAATAGTTACTTGGCTGTGATGG

737 Yes Yan et al., 2001

VIM ForwardReverse

TCTACATGACCGCGTCTGTCTGTGCTTTGACAACGTTCGC

748 Poirel et al., 2000

VIM-1 ForwardReverse

TTATGGAGCAGCAACCGATGTCAAAAGTCCCGCTCCAACGA

920 Yes Yan et al., 2001

VIM-2 ForwardReverse

AAAGTTATGCCGCACTCACCTGCAACTTCATGTTATGCCG

865 Yes Yan et al., 2001

Enzyme Family/ Gene

Primer Primer Sequence (5’-3’) Fragment Size (bp)

Entire coding region

Reference

Class B Metalloenzymes

SPM-1 SPM-1FSPM-1R

CCTACAATCTAACGGCGACCCCTACAATCTAACGGCGACC

650 No Castanheira et al., 2004

GIM-1 GIM-1FGIM-1R

AGAACCTTGACCGAACGCAGACTCATGACTCCTCACGAGG

748 No Castanheira et al., 2004

SIM-1 SIM-1FSIM-1R

TACAAGGGATTCGGCATCGTAATGGCCTGTTCCCATGTG

571 No Lee et al., 2002

NDM-1 ForwardReverse

GGTTTGGCGATCTGGTTTTCCGGAATGGCTCATCACGATC

621 Nordmann et al., 2011

Integron PCR

5’CS3’CS

GGCATCCAAGCAGCAAGAAGCAGACTTGACCTGA

Variable Levesque et al., 1995

Enzyme Family/ Gene

Primer Primer Sequence (5’-3’) Fragment Size (bp)

Entire coding region

Reference

Class D OxacillinasesOXA forward

reverseATG GCA ATC CGA ATC TTC GTTA TCG CGC AGC GTC CGA G

Mohammed Shahid et al., 2012

OXA-23 P5P6

AAGCATGATGAGCGCAAAGAAAAGGCCCATTTATCTCAAA

1,066 Yes Donald et al., 2000

OXA-24 ForwardReverse

GTACTAATCAAAGTTGTGAATTCCCCTAACATGAATTTGT

1023 Yes Afzal-Shah et al., 1998

OXA-69 OXA-69AOXA-69B

CTAATAATTGATCTACTCAAGCCAGTGGATGGATGGATAGATTATC

975 Yes Heritier et al., 2005a

OXA-58 Pre-OXA-58prom+Pre-OXA-58B

TTATCAAAATCCAATCGGC

TAACCTCAAACTTCTAATTC

934 Yes Heritier et al., 2005b

ShewanellaOXA-55

OXA-55/10XA-55/2

CATCTACCTTTAAAATTCCCAGCTGTTCCTGCTTGAGCAC

875 Yes Heritier et al., 2004

Enzyme Family/ Gene

Primer Primer Sequence (5’-3’) Fragment Size (bp)

Entire coding region

Reference

Class D Oxacillinases

OXA-48 OXA-48AOXA-48B

TTGGTGGCATCGATTATCGGGAGCACTTCTTTTGTGATGGC

744 No Poirel et al., 2004

OXA-50 SAS

AATCCGGCGCTCATCCATCGGTCGGCGACTGAGGCGG

869 Yes Girlich et al., 2004a

OXA-60 OXA-60AOXA-60B

AAAGGAGTTGTCTCATGCTGTCTCGAACCTACAGGCGCGCGTCTCACGGTG

353 Yes Girlich et al., 2004b

Multiplex PCR for OxAs in Acinetobacter baumannii

Gene Primer (5’-3’)

OXA-51-like TAATGCTTTGATCGGCCTTG

TGGATTGCACTTCATCTTGGOXA-23-like GATCGGATTGGAGAACCAGA

ATTTCTGACCGCATTTCCATOXA-24-like GGTTAGTTGGCCCCCTTAAA

AGTTGAGCGAAAAGGGGATTOXA-58-like AAGTATTGGGGCTTGTGCTG

CCCCTCTGCGCTCTACATAC

Woodford et al., 2006

Probe based qPCR

Target gene Primer/probe Reference

Sequence 5’ – 3’

Bla KPC & NDM

KPC160F ATTGGCTAAAGGGAAACACGACC Cunningham et al., 2013

KPC160R GTAGACGGCCAACACAAT

NDM1F ATTAGCCGCTGCATTGAT

NDM1R GGCATGTCGAGATAGGAAGT

KPC160fl GAACCGCGGAGTGTATGGCACGG FITC

KPC160iLC610 AAATGACTATGCCGTCGTCTGGCCCACT Red610

NDMfl CAACGGTTTGGCGATCTGGT FITC

NDMiLC670 Red670 TCCGCCAGCTCGCACCG TPO4

Target gene Primer/probe Reference

Sequence 5’ – 3’

Bla KPC & NDM NDM-F

TTGGCGATCTGGTTTTCC Zheng et al., 2103

NDM-R GGTTGATCTCCTGCTTGA

NDM-probe JOE-TGGCAGCACACTTCCTATCTCG- ECLIPSE

KPC-F CGCAACTGTAAGTTACCG

KPC-R CATGCCTGTTGTCAGATA

KPC-probe FAM –CCACTGTGCAGCTCATTCAAGG - ECLIPSE

Bla NDM blaNDM1_F CGC AAC ACA GCC TGA CTT T Ong et al., 2011

blaNDM1_R TCG ATC CCA ACG GTG ATA TT

blaNDM1_P 6FAM-CAA CTT TGG CCC GCT CAA GGT ATT T-BHQ1

LAMP assays for Carbapenemases

Gene Primer Sequence Reference

OX 23 OXA23 F3 GAAGCCATGAAGCTTTCTG Yammato et al., 2015

OXA23 B3 GTATGTGCTAATTGGGAAACA

OXA23 FIP ACCGAAACCAATACGTTTTACTTCTCAGTCCCAGTCTATCAGGA

OXA23 BIP CTGAAATTGGACAGCAGGTTGACTCTACCTCTTGAATAGGCG

OXA23 LF TTTTGCATGAGATCAAGACCGA

OXA23 LB CTGGTTGGTAGGACCATTAAAGGTT

Gene Primer Sequence Reference

NDM & KPC

KPC F3 TCGAACAGGACTTTGGCG Solanki et al., 2013

KPC B3 GGAACCAGCGCATTTTTGCKPC FIP CACAGTGGGAAGCGCTCCTCTTTTGTGTACGCGATGGATACCG

KPC3 BIP TCAAGGGCTTTCTTGCTGCCGTTTTCGTAACGGATGGGTGTGTC

KPC BLP AGCAGCAGGCCGGCTTGCTG

KPC TAACTACAGTTGCGCCTGAGCNDM-1F3 GCATAAGTCGCAATCCCCGNDM-1B3 CTTCCTATCTCGACATGCCGNDM-1FIP GAGATCAACCTGCCGGTCGCTTTTTCCATACCGCCCATCTTGTNDM-1BIP TCTGGGCGGTCTGGTCATCGTTTTTTCCAACGGTTTGATCGTCANDM-1FLP GGTGACTCACGCGCATCAGGNDM-1BLP ACCACCAGCACGCGGCCGCCATC

Gene Primer Sequence(5’-3’) Reference

NDM CJXJ1F3 Forward outer GCATAAGTCGCAATCCCCGC Liu et al., 2012

outerJXJ1B Backward outer GGTTTGATCGTCAGGGATGGC

JXJ1FIP Forward inner CTGGCGGTGGTGACTCACGTTTTGCATGCAGCGCGTCCAC

JXJ1BIP Backward inner CGCGACCGGCAGGTTGATCTTTTGGTCGATACCGCCTGGACC

JXJ1LF1 Loop forward GCATCAGGACAAGATGGGCC

JXJ1LB1 Loop backward TCCAGTTGAGGATCTGGGC

Commercial KitsKPC & NDM surveillance PCR – (Mayo

Medical Laboratories)Hylex MBL ID –Multiplex PCR ELISA – target

genes IMP & VIMHyplex super bug- Multiplex PCR ELISA –

target genes KPC, VIM, NDM, OX 48 –(Amplex diagnostics)