—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 the tyrB 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

Klebsiella pneumoniae, Corynebacterium kutscheri, and Streptococcus pneumoniae are pathogens that cause respiratory disease in laboratory rodents. K. pneumoniae is a gram-negative bacterium that normally inhabits the intestinal tract of rats, mice, and other animals [2]. As an opportunistic pathogen, K. pneumoniae has been re-ported to infect mice [14] and rats [5], and its prevalence is increased in antibiotic-treated nude rats to eliminate

other bacteria [4]. C. kutscheri is a genus of gram-positive bacteria. natural infection with C. kutscheri is usually subclinical, but stresses such as nutritional defi-ciencies and experimental manipulation can induce clinical diseases in the rat and mouse [1]. S. pneumoni-ae is a gram-positive, α-hemolytic, encapsulated diplo-cocci. Clinical signs of S. pneumoniae are uncommon in laboratory rats and mice, although natural outbreaks of this pathogen have been reported [11]. K. pneumoni-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

Exp. Anim. 62(1), 35–40, 2013

©2013 Japanese Association for Laboratory Animal Science

E.-S. JEong, ET AL.36

health monitoring in rodents [12, 13].Bacterial culture is a primary tool used to diagnose

bacterial diseases. However, it is difficult to standardize this method for routine health monitoring compared with PCR-based methods because media, protocols, or cultur-ing conditions may vary greatly among laboratories [11].

We evaluated different bacterial detection methods used by several diagnostic laboratories and searched for primer sequences specific for murine bacteria. Target nucleic acid fragments were amplified by consensus PCR from specific genes of each bacterium. In this study, PCR was used for rapid identification of K. pneumoniae, C. kutscheri, and S. pneumoniae. next, three primer pairs specific for K. pneumoniae, C. kutscheri, and S. pneu-moniae were used in combination for the triplex PCR assays. These assays were developed for simultaneous detection of these three respiratory bacteria in rodents. The triplex PCR assay is a useful and convenient meth-od for the rapid identification of bacterial pathogens in laboratory animals. Furthermore, our assay can be used to easily screen for pathogenic bacterial infections in laboratory animal facilities and monitor the health of laboratory animals.

Materials and Methods

Bacterial strains and DNA preparationThe bacterial strains used in this study are listed in

Table 1. Bacterial strains were grown at 37°C for 48 to 96 h under aerobic or microaerobic conditions. To extract template DnA, we used an AccuPrep genomic DnA

Extraction Kit (Bioneer inc., Daejeon, Republic of Korea) as described in our previous report [7]. Briefly, 500 µl of a lysis solution was added to each microtube and incu-bated overnight at 60°C in a water bath. After 100 µl of isopropanol was added, the samples were mixed by pipet-ting. The lysates were carefully transferred to the upper reservoir of the binding column tube without wetting the rim and then centrifuged at 8,000 rpm for 1 min. Washing solution i and ii were added, and the samples were cen-trifuged at 12,000 rpm for 1 min. The binding column was transferred to a new 1.5 ml tube. one hundred mi-croliters of elution buffer was then added, and the column was allowed to sit for at least 1 min at room temperature. Finally, the samples were centrifuged at 8,000 rpm for 1 min to elute the DnA. A 1 µl aliquot (200 ng/µl) of the eluted DnA was used as the template for PCR. All DnA preparations were stored at −20°C until use.

PCR amplificationDNA was amplified in 20 µl of a PCR mixture contain-

ing 30 mM KCl, 10 mM Tris, 1.5 mM MgCl2, 250 µM of each deoxynucleoside triphosphate (dTTP, dATP, dCTP, and dGTP), 10 pmol of each primer, 1 µl template DnA, and 1 unit of Taq DnA polymerase (Bioneer inc.). The primers used in this study are listed in Table 2. To determine the optimal annealing temperature for the triplex PCR method, a temperature gradient experiment from 55 to 61°C was performed. When the annealing temperature was increased, the band’s intensity was too low (data not shown). The cycling conditions included an initial denaturation for 5 min at 95°C; 35 cycles of 1

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.

TRIPLEX PCR OF RESPIRATORY BACTERIA 37

min at 95°C, 1 min at 55°C, and 1 min at 72°C; and a final extension for 10 min at 72°C. All PCR products were separated on a 2% agarose gel and stained with ethidium bromide.

Experimental infection of mice with K. pneumoniae, C. kutscheri, and S. pneumoniae

Specific pathogen-free BALB/cA mice were obtained at 6 to 7 weeks of age from the Korea Research institute of Bioscience and Biotechnology (KRiBB; Daejeon, Republic of Korea) and maintained in a pathogen-free room with a 12:12 h light-dark cycle. The health status of these mice was monitored routinely by the KRiBB (Daejeon) as recommended by previous reports [11, 12]. These mice were free of murine pathogens including K. pneumoniae, C. kutscheri, and S. pneumoniae. For mouse inoculation, type strain cultures of K. pneumoniae, C. kutscheri, and S. pneumoniae were adjusted to 104, 105, and 107 CFU/30 µl, respectively. To adjust the bacterial concentration, we used the serial dilution and agar plat-ing method as described in our previous report [7]. Six mice were used for a single infection model, and two mice were used for a coinfection study with these three bacteria. The mice were infected with 30 µl of the cul-tures by intratracheal inoculation as described in our previous report [8]. Briefly, mice were anesthetized with intraperitoneal injections of 1.25% Avertin (2,2,2-Tri-bromoethanol) and placed in the dorsal recumbent posi-tion. one of the bacterial suspensions (30 µl) was ad-ministered via sterile 31-gauge needles after exposing the trachea. The skin incision was then closed using surgical staples. The actual dose of bacteria administered was determined by plating serially diluted bacteria on sheep blood agar. infection was established 2 days after inoculation because of clinical signs of ruffled fur, lati-tude, hunched posture and weight loss. The trachea and lungs were collected 2 days after experimental infection for evaluation with the triplex PCR assay. Template DNA

was extracted from trachea and lung tissues using an AccuPrep genomic DnA Extraction Kit (Bioneer inc.) as described in our previous report [7]. A 1 µl aliquot (100 ng/µl) of the eluted DnA was used as the template for PCR. All animal procedures were approved by the institutional Animal Care and Use Committee of Konkuk University.

Results

Specificity and sensitivity of the singleplex PCRWe designed each primer pair to amplify specific tar-

get sequences of the tyrosine aminotransferase (tyrB) gene from K. pneumoniae, gyrase B (gyrB) gene from C. kutscheri, and pneumolysin (ply) gene from S. pneu-moniae; the primer sequences and products size are shown in Table 2. We selected these three genes because the sizes of amplicons do not overlap. To determine singleplex PCR primer specificity for the selected K. pneumoniae-, C. kutscheri-, and S. pneumoniae-specific primers, each primer was tested individually. We ob-served amplified PCR products of 931, 540, and 354 bp for K. pneumoniae, C. kutscheri, and S. pneumoniae, respectively; nonspecific PCR products were not ob-served (Fig. 1A).

The primer pairs did not yield any product from eleven murine pathogens including CAR bacillus, H. hepaticus, M. pulmonis, P. aeruginosa, P. mirabilis, P. multocida, P. pneumotropica, S. aureus, S. epidermidis, S. zooepidemicus, and S. typhimurium (Fig. 1A). These results showed that each primer pair was specific enough to detect its target DnA sequence in the singleplex PCR. Sensitivity of the singleplex PCR was evaluated by 10-fold serial dilutions of DnA isolated from pure cultures of K. pneumoniae, C. kutscheri, and S. pneumoniae type strains. The detection threshold for our singleplex PCR method was 1 pg for C. kutscheri and S. pneumoniae, and 10 pg for K. pneumoniae (Fig. 1B).

Table 2. Primers used in this study

Species Sequences Product size (bp) and target gene

genBank access number 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 studyC. 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 studyS. 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

E.-S. JEong, ET AL.38

Specificity and sensitivity of the triplex PCRThe selected K. pneumoniae-, C. kutscheri-, and S.

pneumoniae-specific primers were tested in combination to determine the triplex PCR specificity. We observed PCR products of 931, 540, and 354 bp for K. pneumoni-ae, C. kutscheri, and S. pneumoniae, respectively (Fig. 2A). The primer pairs did not yield any product when eleven different bacteria that can infect mice and rats were tested. These results demonstrate there was no cross-reactivity in the triplex PCR. The triplex PCR sensitivity was evaluated by 10-fold serial dilutions of DnA isolated from pure cultures of K. pneumoniae, C. kutscheri, and S. pneumoniae type strains (Fig. 2B). The detection thresholds for our triplex PCR method were 10 pg for K. pneumoniae and S. pneumoniae, and 100 pg for C. kutscheri. Each of these bacteria strains could be detected in a mixture of all three bacteria (Fig. 2B).

Evaluation of the triplex PCR for detecting pathogens in experimentally infected mice

The ability of the triplex PCR to detect pathogens in the lung and trachea of experimentally infected mice was evaluated. All kinds of bacteria used in the single infec-tion study were identified in the trachea and lung of experimentally infected mice at 2 days after experimen-tal infection (Fig. 3). in the coinfection study with these three bacteria, the three bacteria strains were simultane-ously identified in the trachea and lung at 2 days after infection using the triplex PCR (Fig. 3).

Discussion

K. pneumoniae, C. kutscheri, and S. pneumoniae are important pathogens that cause respiratory infections in laboratory animals. At present, K. pneumoniae, C.

Fig. 1. Specificity and sensitivity of singleplex PCR for detecting Klebsiella pneumoniae (931 bp), Corynebacterium kutscheri (540 bp), and Streptococcus pneumoniae (354 bp). (A) Specificity of singleplex PCR for detecting K. pneumoniae, C. kutscheri, and S. pneumoniae. M, 100 bp ladder; lane 1, negative control (no template); lane 2, Cilia-associated respiratory bacillus; lane 3, C. kutscheri; lane 4, H. hepaticus; lane 5, K. pneumoniae; lane 6, M. pulmonis; lane 7, P. multocida; lane 8, P. pneumotropica; lane 9, P. aeruginosa; lane 10, S. typhimurium; lane 11, S. pneumoniae; lane 12, P. mirabi-lis; lane 13, S. aureus; lane 14, S. epidermidis; lane 15, S. zooepidemicus. PCR assays were performed with template DnA concentrations (10 ng each). (B) Sensitivity of singleplex PCR for detecting K. pneumoniae, C. kutscheri, and S. pneumoniae. M, 100 bp ladder; lane 1, negative control (no template); lane 2, 200 ng; lane 3, 100 ng; lane 4, 10 ng; lane 5, 1 ng; lane 6, 100 pg ; lane 7, 10 pg; lane 8, 1 pg; lane 9, 100 fg. Con-centrations are for K. pneumoniae, C. kutscheri, and S. pneumoniae, respectively. All PCR products were separated on a 2% agarose gel and stained with ethidium bromide.

TRIPLEX PCR OF RESPIRATORY BACTERIA 39

kutscheri, and S. pneumoniae are considered routine test items in microbiologic monitoring of laboratory rodents in Korea. Also, these bacteria were selected because of their importance for monitoring laboratory animal health in north America and Europe [12, 13]. Conventional diagnosis of infections such as bacteria caused by K.

pneumoniae, C. kutscheri, and S. pneumoniae mainly relies on culture-based testing. These pathogenic bacte-ria are easily grown on culture agar. However, the diag-nostic results from these cultures are usually obtained days or up to a week after sampling. Furthermore, con-ventional bacterial culturing is not always successful in the laboratory because the culturing conditions and methods may be unsuitable for the bacterial species in question [6].

Nucleic acid amplification can overcome these prob-lems and hasten the diagnostic procedures. Rapid diag-nostic techniques can also help reduce the spread of infectious disease. For multiplex PCR, two or more primer pairs are added to one reaction tube, and two or more DNA templates are targeted simultaneously. This is a relatively simple molecular-based method to detect multiple bacterial strains in one PCR reaction. The primer pairs should be specific for the genes of interest, and the PCR products should be of different sizes [9]. in our study, the target nucleic acid fragments were spe-cifically amplified from the tyrB gene of K. pneumoniae, gyrB gene of C. kutscheri, and ply gene of S. pneumoni-ae by consensus PCR. No cross-amplification of the three targets was observed in the eleven bacteria strains that were tested (Cilia-associated respiratory bacillus, H.

Fig. 2. Specificity and sensitivity of triplex PCR for detecting Klebsiella pneumoniae (931 bp), Corynebacterium kutscheri (540 bp), and Streptococcus pneumoniae (354 bp). (A) Specific-ity of triplex PCR for detecting K. pneumoniae, C. kutscheri, and S. pneumoniae. M, 100 bp ladder; lane 1, negative control (no template); lane 2, Cilia-associated respiratory bacillus; lane 3, C. kutscheri; lane 4, H. hepaticus; lane 5, K. pneumoniae; lane 6, M. pulmonis; lane 7, P. multocida; lane 8, P. pneumotropica; lane 9, P. aeruginosa; lane 10, S. typhimurium; lane 11, S. pneumoniae; lane 12, P. mirabilis; lane 13, S. aureus; lane 14, S. epidermidis; lane 15, S. zooepidemicus. PCR assays were performed with template DnA concentrations (10 ng each). (B) Sensitivity of triplex PCR for detection of K. pneumoniae, C. kutscheri, and S. pneumoniae. M, 100 bp ladder; lane 1, negative control (no template); lane 2, 200 ng; lane 3, 100 ng; lane 4, 10 ng; lane 5, 1 ng; lane 6, 100 pg ; lane 7, 10 pg; lane 8, 1 pg; lane 9, 100 fg. Concentrations are for K. pneumoniae, C. kutscheri, and S. pneumoniae, respectively. All PCR products were separated on a 2% agarose gel and stained with ethidium bromide.

Fig. 3. Triplex PCR detection of Klebsiella pneumoniae (931 bp), Corynebacterium kutscheri (540 bp), and Streptococcus pneumoniae (354 bp) in the trachea and lung of experi-mentally infected mice. M, 100 bp ladder; lane 1, negative control (no template); lanes 2–3, mice infected with K. pneumoniae; lanes 4–5, mice infected with C. kutscheri; lanes 6–7, mice infected with S. pneumoniae; lanes 8–9, mice coinfected with K. pneumoniae, C. kutscheri, and S. pneumoniae. PCR assays were performed with template concentrations of up to 100 ng. All PCR products were separated on a 2% agarose gel and stained with ethidium bromide.

E.-S. JEong, ET AL.40

hepaticus, M. pulmonis, P. aeruginosa, S. typhimurium, P. mirabilis, P. multocida, P. pneumotropica, S. aureus, S. epidermidis and S. zooepidemicus). overall, triplex PCR showed excellent specificity for detecting patho-genic specimens that cause diseases in laboratory mice and rats (Fig. 2A).

The use of singleplex PCR assays for detecting S. pneumoniae in sputum and throat samples from pneu-monia patients has been reported [3, 10]. Recently, mul-tiplex PCR was used to detect Staphylococcus aureus (femA and mecA genes), K. pneumoniae (mdh gene), P. aeruginosa (oprL gene), Acinetobacter baumannii (gltA gene), and Escherichia coli (phoA gene) [15]. To the best of our knowledge, simultaneous detection of K. pneu-moniae, C. kutscheri, and S. pneumoniae by triplex PCR has not been previously reported.

The limits of detection for the triplex PCR assay ranged from 10 to 100 pg of DnA (Fig. 2B). We exam-ined the applicability of our triplex PCR assay using experimentally infected mice. As shown in Fig. 3, the three bacteria strains used to inoculate the mice were successfully identified in the trachea and lung. Our re-sults demonstrate that this method could be used for the accurate and rapid diagnosis of K. pneumoniae, C. kutscheri, and S. pneumoniae infections.

in summary, our triplex PCR assay is simple, fast, sensitive, and specific, and can simultaneously detect K. pneumoniae, C. kutscheri, and S. pneumoniae. We also found that a single triplex PCR test can simultaneously detect these three common bacterial pathogens in ex-perimentally infected mice. This assay is simpler and faster than conventional culture-based methods. our results demonstrate that the triplex PCR assay could be used to detect pathogenic bacterial infections in labora-tory mice and rats and serve as a method for monitoring the health of laboratory animal populations.

Acknowledgment

This paper was supported by Konkuk University in 2012.

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