PCR based Detection of Carbapenamases: Different primers and their utility
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Carbapenems are Wide spectrum, β lactam class of antibiotic drug of last resort available as: imipenam, meropenam, doripenam and ertapenam. However, resistance to these drugs is emerging and may be mediated through: Efflux mechanism, Loss of porins, Change/ alterations in PBPs and specific β-lactamases - commonly known as carbapenamases.
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PCR based Detection of Carbapenamases: Different primers and their utility
- 1. PCR based detection of Carbapenamases: Different primers and their utility Philma Glora. M & Bhoj R Singh Division of Epidemiology Indian veterinary Research Institute, Izatnagar- 243 122, India
- 2. Carbapenams Wide spectrum, β lactam class of antibiotics Drug 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.
- 3. Class A Carbapenemases Serine residue at active site of enzyme Plasmid/chromosome Generally inhibited by clavulanic acid Examples Chromosomally encoded Plasmid encoded SME (Serratia marcescens enzyme) GES (Guiana extended spectrum) NMC (Non-metalloenzyme carbapenamase) KPC (Klebsiella pneumoniae carbapenamase) IMI (Imipenam hydrolyzing β-lactamase)
- 4. Class B Carbapenemases Zn at enzyme active site Inhibited by EDTA Located in integrons/plasmid Examples VIM (Verona integron encoded β-lactamase) SMP (Sao Paulo metallo β-lactamase) NDM1 (New Delhi metallo β-lactamase) IMP (Active on Imipenam)
- 5. Class C Carbapenemases Low potential of carbapenam resistance when combined with efflux mechanism over expression pYMG-1 Class D Carbapenemases Oxacillin hydrolysis Serine at active site of enzyme Plasmid encoded Common in Acinetobacter spp. Poorly inhibited by EDTA and clavulanic acid Examples-OX-23, OX-48
- 6. Class Producer Organisms Hydrolysis profile of the enzymes Penicillin Early cephalosporins Extended Spectrum cephalosporins Aztreonam Carbapenams A Member of Enterobacteri aceae, Pseudomonas, Klebsiella + + + + (except GES) + B Pseudomonas, members of Enterobacteri aceae, Acinetobacter + + + - + D Acinetobacter + + ± - ±
- 7. Detection of Carbapenemases withPCR Nucleic 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.
- 8. Enzyme Family/ Gene Primer Primer Sequence (5’-3’) Fragment Size (bp) Entire coding region Reference Class A Carbapenameses NMC NMC1 NMC4 GCATTGATATACCTTTAGCAGAGA CGGTGATAAAATCACACTGAGCATA 2,158 Yes Radice et al., 2004 SME IRS-5 IRS-6 AGATAGTAAATTTTATAG CTCTAACGCTAATAG 1,138 Yes Queenan et al., 2000 IMI IMI-A IMI-B ATAGCCATCCTTGTTTAGCTC TCTGCGATTACTTTATCCTC 818 No Aubron et al., 2004 KPC KPC forward KPC reverse ATGTCACTGTATCGCCGTCT TTTTCAGAGCCTTACTGCCC 893 Yes Bradford et al., 2004 GES GES-C GES-D GTTTTGCAATGTGCTCAACG TGCCATAGCAATAGGCGTAG 371 No Weldhagen et al., 2004
- 9. Enzyme Family/ Gene Primer Primer Sequence (5’-3’) Fragment Size (bp) Entire coding region Reference Class B Metalloenzymes IMP Forward Reverse CTACCGCAGCAGAGTCTTTG AACCAGTTTTGCCTTACCAT 587 Senda et al., 1996 IMP-1 Forward Reverse TGAGCAAGTTATCTGTATTC TTAGTTGCTTGGTTTTGATG 740 Yes Yan et al., 2001 IMP-2 Forward Reverse GGCAGTCGCCCTAAAACAAA TAGTTACTTGGCTGTGATGG 737 Yes Yan et al., 2001 VIM Forward Reverse TCTACATGACCGCGTCTGTC TGTGCTTTGACAACGTTCGC 748 Poirel et al., 2000 VIM-1 Forward Reverse TTATGGAGCAGCAACCGATGT CAAAAGTCCCGCTCCAACGA 920 Yes Yan et al., 2001 VIM-2 Forward Reverse AAAGTTATGCCGCACTCACC TGCAACTTCATGTTATGCCG 865 Yes Yan et al., 2001
- 10. Enzyme Family/ Gene Primer Primer Sequence (5’-3’) Fragment Size (bp) Entire coding region Reference Class B Metalloenzymes SPM-1 SPM-1F SPM-1R CCTACAATCTAACGGCGACC CCTACAATCTAACGGCGACC 650 No Castanheira et al., 2004 GIM-1 GIM-1F GIM-1R AGAACCTTGACCGAACGCAG ACTCATGACTCCTCACGAGG 748 No Castanheira et al., 2004 SIM-1 SIM-1F SIM-1R TACAAGGGATTCGGCATCG TAATGGCCTGTTCCCATGTG 571 No Lee et al., 2002 NDM-1 Forward Reverse GGTTTGGCGATCTGGTTTTC CGGAATGGCTCATCACGATC 621 Nordmann et al., 2011 Integron PCR 5’CS 3’CS GGCATCCAAGCAGCAAG AAGCAGACTTGACCTGA Variable Levesque et al., 1995
- 11. Enzyme Family/ Gene Primer Primer Sequence (5’-3’) Fragment Size (bp) Entire coding region Reference Class D Oxacillinases OXA forward reverse ATG GCA ATC CGA ATC TTC G TTA TCG CGC AGC GTC CGA G Mohammed Shahid et al., 2012 OXA-23 P5 P6 AAGCATGATGAGCGCAAAG AAAAGGCCCATTTATCTCAAA 1,066 Yes Donald et al., 2000 OXA-24 Forward Reverse GTACTAATCAAAGTTGTGAA TTCCCCTAACATGAATTTGT 1023 Yes Afzal-Shah et al 1998 OXA-69 OXA-69A OXA-69B CTAATAATTGATCTACTCAAG CCAGTGGATGGATGGATAGATTATC 975 Yes Heritier et al., 2005a OXA-58 Pre-OXA- 58prom+ Pre-OXA- 58B TTATCAAAATCCAATCGGC TAACCTCAAACTTCTAATTC 934 Yes Heritier et al., 2005b Shewanella OXA-55 OXA-55/1 0XA-55/2 CATCTACCTTTAAAATTCCC AGCTGTTCCTGCTTGAGCAC 875 Yes Heritier et al., 2004
- 12. Enzyme Family/ Gene Primer Primer Sequence (5’-3’) Fragment Size (bp) Entire coding region Reference Class D Oxacillinases OXA-48 OXA-48A OXA-48B TTGGTGGCATCGATTATCGG GAGCACTTCTTTTGTGATGGC 744 No Poirel et al., 2004 OXA-50 S AS AATCCGGCGCTCATCCATC GGTCGGCGACTGAGGCGG 869 Yes Girlich et al., 2004a OXA-60 OXA-60A OXA-60B AAAGGAGTTGTCTCATGCTGTCTCG AACCTACAGGCGCGCGTCTCACGGTG 353 Yes Girlich et al., 2004b
- 13. Multiplex PCR for OxAs in Acinetobacter baumannii Gene Primer (5’-3’) OXA-51-like TAATGCTTTGATCGGCCTTG TGGATTGCACTTCATCTTGG OXA-23-like GATCGGATTGGAGAACCAGA ATTTCTGACCGCATTTCCAT OXA-24-like GGTTAGTTGGCCCCCTTAAA AGTTGAGCGAAAAGGGGATT OXA-58-like AAGTATTGGGGCTTGTGCTG CCCCTCTGCGCTCTACATAC Woodford et al., 2006
- 14. 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
- 15. 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
- 16. LAMP assays for Carbapenemases Gene Primer Sequence Reference OX 23 OXA23 F3 GAAGCCATGAAGCTTTCTG Yammato et al., 2015 OXA23 B3 GTATGTGCTAATTGGGAAACA OXA23 FIP ACCGAAACCAATACGTTTTACTTCTCAGTCCCAGTCTATC AGGA OXA23 BIP CTGAAATTGGACAGCAGGTTGACTCTACCTCTTGAATA GGCG OXA23 LF TTTTGCATGAGATCAAGACCGA OXA23 LB CTGGTTGGTAGGACCATTAAAGGTT
- 17. Gene Primer Sequence Reference NDM & KPC KPC F3 TCGAACAGGACTTTGGCG Solanki et al., 2013 KPC B3 GGAACCAGCGCATTTTTGC KPC FIP CACAGTGGGAAGCGCTCCTCTTTTGTGTACGCGATGGATACCG KPC3 BIP TCAAGGGCTTTCTTGCTGCCGTTTTCGTAACGGATGGGTGTGTC KPC BLP AGCAGCAGGCCGGCTTGCTG KPC TAACTACAGTTGCGCCTGAGC NDM-1F3 GCATAAGTCGCAATCCCCG NDM-1B3 CTTCCTATCTCGACATGCCG NDM-1FIP GAGATCAACCTGCCGGTCGCTTTTTCCATACCGCCCATCTTGT NDM-1BIP TCTGGGCGGTCTGGTCATCGTTTTTTCCAACGGTTTGATCGTCA NDM-1FLP GGTGACTCACGCGCATCAGG NDM-1BLP ACCACCAGCACGCGGCCGCCATC
- 18. Gene Primer Sequence(5’-3’) Reference NDM CJXJ1F3 Forward outer GCATAAGTCGCAATCCCCGC Liu et al., 2012 outerJXJ1B Backward outer GGTTTGATCGTCAGGGATGGC JXJ1FIP Forward inner CTGGCGGTGGTGACTCACGTTTTGCATGCAGCGC GTCCAC JXJ1BIP Backward inner CGCGACCGGCAGGTTGATCTTTTGGTCGATACCG CCTGGACC JXJ1LF1 Loop forward GCATCAGGACAAGATGGGCC JXJ1LB1 Loop backward TCCAGTTGAGGATCTGGGC
- 19. Commercial Kits KPC & NDM surveillance PCR – (Mayo Medical Laboratories) Hylex MBL ID –Multiplex PCR ELISA – target genes IMP & VIM Hyplex super bug- Multiplex PCR ELISA – target genes KPC, VIM, NDM, OX 48 –(Amplex diagnostics)
Editor's Notes
- DUPLEX TAQMAN TAQMAN-PROBE BASED FRET 2)