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

• Published in: The Journal of molecular diagnostics : JMD Vol. 11; no. 5; pp. 464 - 471
• Main Authors: Hartman, Laurie J, Selby, Edward B, Whitehouse, Chris A, Coyne, Susan R, Jaissle, James G, Twenhafel, Nancy A, Burke, Robin L, Kulesh, David A
• Format: Journal Article
• Language: English
• Published: United States ASIP 01-09-2009; American Society for Investigative Pathology
• Subjects: Animals; Bacterial Proteins - genetics; Cercopithecus aethiops; Klebsiella Infections - diagnosis; Klebsiella Infections - microbiology; Klebsiella pneumoniae - genetics; Klebsiella pneumoniae - isolation & purification; Phenotype; Polymerase Chain Reaction - methods; Primates; Regular; Viscosity
• ISSN: 1525-1578; 1943-7811
• DOI: 10.2353/jmoldx.2009.080136

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.