276. Using Molecular Diagnostics to Decipher the Genetic Basis of Antibiotic Resistance in Clinical Isolates of Gram-Negative Bacteria
Session: Poster Abstract Session: Diagnostic Microbiology; Novel Molecular Methods
Thursday, October 3, 2013
Room: The Moscone Center: Poster Hall C
Background: The development of rapid and accurate molecular diagnostics is a promising strategy to combat the challenge of multidrug-resistant (MDR) Gram-negative bacteria. Once robust clinical links are established, molecular diagnostics have the potential to expedite targeted therapy, thereby improving clinical outcome and reducing unnecessary use of antibiotics. Here, we evaluated the utility of two novel molecular diagnostic platforms, one based on PCR combined with electro-spray ionization mass spectrometry (PCR/ESI-MS), and the other using DNA microarrays.

Methods: We selected 50 clinical bacterial isolates previously identified as Klebsiella pneumoniae (n=22), Escherichia coli (n=11), Pseudomonas aeruginosa (n=2), Enterobacter spp. (n=5), and 10 other genera. These isolates are known to express a wide variety of beta-lactamase (bla) genes, such as SHV/TEM ESBLs and non-ESBLs, OXA, KPC, CMY, CTX-M, ACT, MIR, DHA, NDM, VIM and IMP. PCR/ESI-MS (Ibis Biosciences) and DNA microarray (CheckPoints CT103 microarray kit) were used to identify the genetic determinants of bla mediated resistance per isolate. Additionally, PCR/ESI-MS provided bacterial species identification. The data from both methodologies were compared, and discrepant results examined.

Results: Both platforms achieved reproducibility within each technology as well as when compared to one another. Detection of carbapenemase genes, including 15 KPCs, 6 NDMs, 4 VIMs, 2 OXA-48s, and 1 IMP was 100% accurate with both methods. Additionally, in all but 7 isolates, a SHV/TEM ESBL and/or non-ESBL genes were detected, along with 27 CTX-M and 11 AmpC genes. Both platforms were able to detect a complex background of 3-4 bla genes per isolate, with various combinations. After discrepancy resolution, overall agreement was 92% (46/50 isolates had perfect bla gene agreement) between methods. In four samples, bacterial species identification with PCR/ESI-MS did not match the original identification and 2 samples contained mixed cultures.

Conclusion: PCR/ESI-MS and DNA microarrays accurately detected a diverse variety of bla genes in Gram-negative bacteria. Use of these methods in clinical trials may translate into diagnostic tools for the early and accurate identification of MDR organisms, and the implementation of precisely targeted antibiotic therapy.

Andrea M. Hujer, BS1, Kristine M. Hujer, BS1, Thomas Hall, PhD2, Christine Marzan, PhD2, Rangarajan Sampath, PhD2, David J. Ecker, PhD2, T. Nicholas Domitrovic, BA3, Liang Chen, PhD4, Federico Perez, MD3, Barry Kreiswirth, PhD4, Vance Fowler, MD5, Henry F. Chambers, MD6 and Robert A. Bonomo, MD3, (1)Case Western Reserve University, Cleveland, OH, (2)Ibis Biosciences, Carlsbad, CA, (3)Louis Stokes Cleveland Veterans Affairs Medical Center, Cleveland, OH, (4)Public Health Research Institute Center, University of Medicine and Dentistry of New Jersey, Newark, NJ, (5)Duke University Medical Center, Durham, NC, (6)UCSF - San Francisco General Hospital, San Francisco, CA


A. M. Hujer, None

K. M. Hujer, None

T. Hall, Ibis Biosciences: Employee, Salary

C. Marzan, Ibis Biosciences: Employee, Salary

R. Sampath, Ibis Biosciences: Employee, Salary

D. J. Ecker, Ibis Biosciences: Employee, Salary

T. N. Domitrovic, None

L. Chen, None

F. Perez, None

B. Kreiswirth, None

V. Fowler, Merck: Investigator, Research support

H. F. Chambers, None

R. A. Bonomo, None

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