Techniques for the Rapid Detection of KPC Gene Isolated from Bacteria found in Sputum Samples using PCR Amplification.

Rosmely Hernandez Department of Biology UNA Some bacteria produce carbapenemase from a KPC gene on genomic or plasmid DNA.

DNA RNA protein KPC gene KPC RNA carbapenamase Mechanism of action of carbapenemase 2 2 1 R 1 R R R 3 3 Carbapenemase - R R O Carbapenem N Hydrolysis N O O O O - HO O Site of beta lactamase action

O O NH S NH S Carbapenemase 2 2 R R - CH2 N O 1 Hydrolysis N Cephalosporin O R O O - HO O O Site of beta lactamase action Families of antibiotics affected by carbapenemases

Penicillins Carbapenems Monobactams Cephalosporins Penicillin G Imipenem Aztreonam Cefacetrile Penicillin V Meropenem Cefadroxil Ampicillin Ertapenem Cefalexin Amoxicillin Doripenem Cefaloglycin Methicillin Cefalonium Oxacillin Cefaloridine Piperacillin Cefalotin

Importance of identifying KPC+ bacteria Experimental design

I. Design primers for PCR of KPC gene II. Isolate DNA from KPC+ and KPC- bacteria III. Perform PCR for KPC gene and a control gene IV. Electrophoretic analysis of PCR products V. Gel extraction and DNA sequencing to confirm KPC gene detection Primer Design for PCR Klebsiella pneumoniae class A carbapenemase coding sequence NCBI Accession No. NC_014312.1

1 atgtcactgt atcgccgtct agttctgctg tcttgtctct catggccgct ggctggcttt 61 tctgccaccg cgctgaccaa cctcgtcgcg gaaccattcg ctaaactcga acaggacttt 121 ggcggctcca tcggtgtgta cgcgatggat accggctcag gcgcaactgt aagttaccgc 181 gctgaggagc gcttcccact gtgcagctca ttcaagggct ttcttgctgc cgctgtgctg 241 gctcgcagcc agcagcaggc cggcttgctg gacacaccca tccgttacgg caaaaatgcg 301 ctggttccgt ggtcacccat ctcggaaaaa tatctgacaa caggcatgac ggtggcggag 361 ctgtccgcgg ccgccgtgca atacagtgat aacgccgccg ccaatttgtt gctgaaggag 421 ttgggcggcc cggccgggct gacggccttc atgcgctcta tcggcgatac cacgttccgt 481 ctggaccgct gggagctgga gctgaactcc gccatcccag gcgatgcgcg cgatacctca 541 tcgccgcgcg ccgtgacgga aagcttacaa aaactgacac tgggctctgc actggctgcg 601 ccgcagcggc agcagtttgt tgattggcta aagggaaaca cgaccggcaa ccaccgcatc 661 cgcgcggcgg tgccggcaga ctgggcagtc ggagacaaaa ccggaacctg cggagtgtat 721 ggcacggcaa atgactatgc cgtcgtctgg cccactgggc gcgcacctat tgtgttggcc 781 gtctacaccc gggcgcctaa caaggatgac aagcacagcg aggccgtcat cgccgctgcg 841 gctagactcg cgctcgaggg attgggcgtc aacgggcagt aa

68 ˚C improves specificity Isolation of DNA from KPC+ and KPC- bacteria

Handling of KPC+ bacteria

- Why should we be very careful?

- Precautions during DNA isolation

-Gloves, goggles, lab coat, and face mask Electrophoretic Analysis of PCR Template None KPC+ KPC+ KPC- KPC- gDNA gDNA gDNA gDNA

Primers None rDNA KPC rDNA KPC

DNA ladder, bp Band sizes, bp

1500 1484 1000 882 750 500 250 BLAST of PCR product sequence to KPC gene sequence

PCR KPC

Identity- 99% Next steps

Isolate genomic and plasmid DNA from resistant sputum isolates

Perform PCR

Identify whether isolate is KPC+ or KPC- Conclusion

PCR may be used to detect KPC+ bacteria and aid in treatment of infections. Acknowledgments

BBB Research Grant

Dept. of Biology, University of North Alabama

Dr. Tina Hubler and Dr. Lisa Ann Blankinship

Baker, G., Smith, J.J., Cowan, D.A. (2003). Review and re-analysis of Domain-specific 16S primers, Journal of Microbiological Methods 55 (3): 541-555. doi:10.1016/j.mimet.2003.08.009