Exp. Anim. 62(1), 35–40, 2013 —Original— Rapid Identification of Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae Using Triplex Polymerase Chain Reaction in Rodents Eui-Suk JEONG1), Kyoung-Sun Lee1,2), Seung-Ho HEO1,3), Jin-Hee SEO1), and Yang-Kyu CHOI1) 1)Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701, Republic of Korea 2)Osong Medical Innovation Foundation Laboratory Animal Center, 186 Osong Saengmyung-Ro, Gangoe-myeon, Cheongwon-gun, Chungbuk 363-951, Republic of Korea 3)Asan Institute for Life Sciences, University of Ulsan College of Medicine, 388–1 Pungnap-2-dong, Songpa-gu Seoul 138-736, Republic of Korea Abstract: Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are important pathogens that cause respiratory infections in laboratory rodents. In this study, we used species-specific triplex PCR analysis to directly detect three common bacterial pathogens associated with respiratory diseases. Specific targets were amplified with conventional PCR using thetyrB gene from K. pneumoniae, gyrB gene from C. kutscheri, and ply gene from S. pneumoniae. Our primers were tested against purified DNA from another eleven murine bacteria to determine primer specificity. Under optimal PCR conditions, the triplex assay simultaneously yielded a 931 bp product from K. pneumoniae, a 540 bp product from C. kutscheri, and a 354 bp product from S. pneumoniae. The triplex assay detection thresholds for pure cultures were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. All three bacteria were successfully identified in the trachea and lung of experimentally infected mice at the same time. Our triplex PCR method can be used as a useful method for detecting pathogenic bacterial infections in laboratory rodents. Key words: Corynebacterium kutscheri, Klebsiella pneumoniae, Streptococcus pneumoniae, triplex PCR Introduction other bacteria [4]. C. kutscheri is a genus of Gram- positive bacteria. Natural infection with C. kutscheri is Klebsiella pneumoniae, Corynebacterium kutscheri, usually subclinical, but stresses such as nutritional defi- and Streptococcus pneumoniae are pathogens that cause ciencies and experimental manipulation can induce respiratory disease in laboratory rodents. K. pneumoniae clinical diseases in the rat and mouse [1]. S. pneumoni- is a Gram-negative bacterium that normally inhabits the ae is a Gram-positive, α-hemolytic, encapsulated diplo- intestinal tract of rats, mice, and other animals [2]. As cocci. Clinical signs of S. pneumoniae are uncommon an opportunistic pathogen, K. pneumoniae has been re- in laboratory rats and mice, although natural outbreaks ported to infect mice [14] and rats [5], and its prevalence of this pathogen have been reported [11]. K. pneumoni- is increased in antibiotic-treated nude rats to eliminate ae, C. kutscheri, and S. pneumoniae are all used for (Received 1 June 2012 / Accepted 6 September 2012) Address corresponding: Y.-K. Choi, Department of Laboratory Animal Medicine, College of Veterinary Medicine, Konkuk University, 1 Hwayang- dong, Gwangjin-gu, Seoul 143-701, Republic of Korea ©2013 Japanese Association for Laboratory Animal Science 36 E.-S. JEONG, ET AL. Table 1. Bacteria strains used in this study Bacteria Strains Supply Cultivation Klebsiella pneumoniae ATCC 13883Ta) KRIBB 5% sheep blood Corynebacterium kutscheri ATCC 15677Ta) ATCC 5% sheep blood Streptococcus pneumoniae ATCC 33400Ta) ATCC 5% sheep blood Helicobacter hepaticus ATCC 51448Ta) KRIBB 5% sheep blood Mycoplasma pulmonis ATCC 19612Ta) ATCC Mycoplasma agar (Oxoid, Hampshire, UK) Pasteurella multocida ATCC 43137Ta) ATCC 5% sheep blood Pasteurella pneumotropica ATCC 35149Ta) KRIBB 5% sheep blood Pseudomonas aeruginosa ATTC 10145Ta) KRIBB Cetrimide (Merck, Darmstadt, Germany) Salmonella typhimurium ATCC 13311Ta) KRIBB DHL (Merck) Cilia-associated respiratory bacillus CBM strain KRIBB –b) Proteus mirabilis KCTC 2566 KTCC 5% sheep blood Staphylococcus aureus E86-13 KRIBB Vogel-Johnson (Merck) Staphylococcus epidermidis KCTC 1917 KTCC 5% sheep blood Streptococcus zooepidemicus F921-4 KRIBB 5% sheep blood Abbreviations: KRIBB, Korea Research Institute of Bioscience and Biotechnology (Daejeon, Republic of Korea); ATCC, American Type Culture Collection (Manassas, VA, USA); KTCC, Korean Collection for Type Cultures (Daejeon, Republic of Korea). a) Type strain. b) DNA obtained from KRIBB. health monitoring in rodents [12, 13]. Extraction Kit (Bioneer Inc., Daejeon, Republic of Korea) Bacterial culture is a primary tool used to diagnose as described in our previous report [7]. Briefly, 500µ l of bacterial diseases. However, it is difficult to standardize a lysis solution was added to each microtube and incu- this method for routine health monitoring compared with bated overnight at 60°C in a water bath. After 100 µl of PCR-based methods because media, protocols, or cultur- isopropanol was added, the samples were mixed by pipet- ing conditions may vary greatly among laboratories [11]. ting. The lysates were carefully transferred to the upper We evaluated different bacterial detection methods reservoir of the binding column tube without wetting the used by several diagnostic laboratories and searched for rim and then centrifuged at 8,000 rpm for 1 min. Washing primer sequences specific for murine bacteria. Target solution I and II were added, and the samples were cen- nucleic acid fragments were amplified by consensus PCR trifuged at 12,000 rpm for 1 min. The binding column from specific genes of each bacterium. In this study, PCR was transferred to a new 1.5 ml tube. One hundred mi- was used for rapid identification of K. pneumoniae, C. croliters of elution buffer was then added, and the column kutscheri, and S. pneumoniae. Next, three primer pairs was allowed to sit for at least 1 min at room temperature. specific for K. pneumoniae, C. kutscheri, and S. pneu- Finally, the samples were centrifuged at 8,000 rpm for 1 moniae were used in combination for the triplex PCR min to elute the DNA. A 1 µl aliquot (200 ng/µl) of the assays. These assays were developed for simultaneous eluted DNA was used as the template for PCR. All DNA detection of these three respiratory bacteria in rodents. preparations were stored at −20°C until use. The triplex PCR assay is a useful and convenient meth- od for the rapid identification of bacterial pathogens in PCR amplification laboratory animals. Furthermore, our assay can be used DNA was amplified in 20µ l of a PCR mixture contain- to easily screen for pathogenic bacterial infections in ing 30 mM KCl, 10 mM Tris, 1.5 mM MgCl2, 250 µM laboratory animal facilities and monitor the health of of each deoxynucleoside triphosphate (dTTP, dATP, laboratory animals. dCTP, and dGTP), 10 pmol of each primer, 1 µl template DNA, and 1 unit of Taq DNA polymerase (Bioneer Inc.). Materials and Methods The primers used in this study are listed in Table 2. To determine the optimal annealing temperature for the Bacterial strains and DNA preparation triplex PCR method, a temperature gradient experiment The bacterial strains used in this study are listed in from 55 to 61°C was performed. When the annealing Table 1. Bacterial strains were grown at 37°C for 48 to temperature was increased, the band’s intensity was too 96 h under aerobic or microaerobic conditions. To extract low (data not shown). The cycling conditions included template DNA, we used an AccuPrep Genomic DNA an initial denaturation for 5 min at 95°C; 35 cycles of 1 TRIPLEX PCR OF RESPIRATORY BACTERIA 37 Table 2. Primers used in this study Product size (bp) and GenBank access number Species Sequences target gene and reference K. pneumoniae 5’ GGC TGT ACT ACA ACG ATG AC 3’ 931 (101–1,031) AF074934.1 3’ TTG AGC AGG TAA TCC ACT TTG 5’ tyrB Present study C. kutscheri 5’ CGT GAT GGC CAT CTT TGG TT 3’ 540 (91–630) AB014265.1 3’ AAT CGT ATT AGC AAA GGT ATG C 5’ gyrB Present study S. pneumoniae 5’ GTG ATA TTT CTG TAA CAG CTA CC 3’ 354 (173–526) GU968411.1 3’ GAG AAT TCC CTG TCT TTT CAA A 5’ ply Present study min at 95°C, 1 min at 55°C, and 1 min at 72°C; and a was extracted from trachea and lung tissues using an final extension for 10 min at 72°C. All PCR products AccuPrep Genomic DNA Extraction Kit (Bioneer Inc.) were separated on a 2% agarose gel and stained with as described in our previous report [7]. A 1 µl aliquot ethidium bromide. (100 ng/µl) of the eluted DNA was used as the template for PCR. All animal procedures were approved by the Experimental infection of mice with K. pneumoniae, C. Institutional Animal Care and Use Committee of Konkuk kutscheri, and S. pneumoniae University. Specific pathogen-free BALB/cA mice were obtained at 6 to 7 weeks of age from the Korea Research Institute Results of Bioscience and Biotechnology (KRIBB; Daejeon, Republic of Korea) and maintained in a pathogen-free Specificity and sensitivity of the singleplex PCR room with a 12:12 h light-dark cycle. The health status We designed each primer pair to amplify specific tar- of these mice was monitored routinely by the KRIBB get sequences of the tyrosine aminotransferase (tyrB) (Daejeon) as recommended by previous reports [11, 12]. gene from K. pneumoniae, gyrase B (gyrB) gene from These mice were free of murine pathogens including K. C. kutscheri, and pneumolysin (ply) gene from S. pneu- pneumoniae, C. kutscheri, and S. pneumoniae. For mouse moniae; the primer sequences and products size are inoculation, type strain cultures of K. pneumoniae, C. shown in Table 2. We selected these three genes because kutscheri, and S. pneumoniae were adjusted to 104, 105, the sizes of amplicons do not overlap. To determine and 107 CFU/30 µl, respectively. To adjust the bacterial singleplex PCR primer specificity for the selected K. concentration, we used the serial dilution and agar plat- pneumoniae-, C.