Z-015. Rapid Real-Time PCR for Detection of Klebsiella pneumoniae and the rmpA Gene Target responsible for the Hypermucoviscosity Phenotype: Comparison with Culture for the Screening of African Green monkeys

E. Selby, L. Hartman, C. Whitehouse, S. Coyne, J. Jaissle, N. Twenhafel, R. Burke, D. Kulesh;
USAMRIID, Fort Detrick, MD.

Background: We previously demonstrated that rmpA+ invasive Klebsiella pneumoniae can be a cause of serious disease in African green monkeys (AGM). To more rapidly screen AGM for this infection, we developed two real-time PCR assays. The first assay was designed to target the chromosomal hemolysin (khe) gene known to be present in all strains of K. pneumoniae. The second assay targeted the rmpA gene, a presumed virulence factor gene which is known to correlate with the hypermucoid phenotype of invasive strains of K. pneumoniae. Both assays were compared to routine plate culture of oral and necropsy swabs from AGM. Methods: Primer Express 2 (ABI) was used with the K. pneumoniae khe (Accession: AF293352.1) and rmpA (Accession: AB289644) genes to generate a series of primers and TaqMan-MGB probes specific for each sequence. Several AGM from the USAMRIID colony that came in contact with an animal previously infected with rmpA+ K. pneumoniae were tested from dual swabs of throats and several tissues after necropsy. One swab was processed for routine microbiology culture by plating on 5% sheep blood agar, chocolate agar and MacConkey agar. A second swab was extracted for DNA using the QIAmp DNA Mini Kit (Qiagen). All extracted DNAs and K. pneumoniae-positive cultures were tested with the khe assay with all khe+ samples also tested with the rmpA assay. Results: The TaqMan-MGB assays were validated to be specific for each gene. Testing of AGM #V513 resulted in a positive test for khe and rmpA. Subsequent testing of 45 AGM resulted in six potential K. pneumoniae colonies and 15 khe+ tests by real-time PCR. No khe+ animal was found to also be positive for rmpA. The six bacterial colonies were extracted and re-analyzed resulting in five khe+/rmpA- tests with the other colony determined to be a Pseudomonas species (khe-/rmpA-). Conclusions: We developed two real-time PCR assays which specifically detect the K. pneumoniae khe and rmpA genes. AGM testing at USAMRIID demonstrated the ability and sensitivity to screen both live and necropsied animals for the presence of K. pneumoniae and to determine if it is the hypermucoid variant by real time PCR.