Published online before print July 30, 2009

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Copyright © 2009 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2009.080136

Accepted for publication April 15, 2009.

Article

Rapid Real-Time PCR Assays for Detection of Klebsiella pneumoniae with the rmpA or magA Genes Associated with the Hypermucoviscosity Phenotype. Screening of Nonhuman Primates

Laurie J. Hartman^*, Edward B. Selby^*, Chris A. Whitehouse^*, Susan R. Coyne^*, James G. Jaissle^*, Nancy A. Twenhafel, Robin L. Burke, and David A. Kulesh^*@

From the Diagnostic Systems Division,* Veterinary Pathology, and Veterinary Medicine, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, Maryland

^@ To whom correspondence should be addressed. E-mail: david.kulesh{at}amedd.army.mil.

	Abstract

The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA+ K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA+ and magA+ HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific TaqMan/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe+ samples, five were culture-positive, but none were HMV "string test"-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA+ and 15 were magA+. Non-human primate testing at the U.S. Army Medical Research Institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype.