PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis

PCR-dot blot hybridization based on the neuraminidase-encoding gene is useful for detection of Bacteroides fragilis

Tomomi Kuwahara, Haruyuki Nakayama, Tsuyosi Miki, Keiko Kataoka, Hideki Arimochi, and Yoshinari Ohnishi

Department of Bacteriology, The University of Tokushima School of Medicine, Tokushima, Japan

Abstract: Bacteroides fragilis is a Gram-negative obligate anaerobe frequently isolated from clinical specimens and sometimes causes severe septicemia in compromised hosts. Increasing interest has been shown in the enterotoxigenicity and drug resistance of B. fragilis in the field of medical microbiology. We previously reported rapid detection of this anaerobe by nested PCR targeting a neuraminidase-encoding gene nanH. In the present study, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1,which is specific for nanH of B. fragilis, and we combined the hybridization assay using NH1with the nanH-PCR to detect this anaerobe in a bacteremia model mice. In the specificity test, the oligonucleotide probe, NH1, hybridized only to amplification products from B. fragilis. PCR-dot blot hybridization based on nanH enabled detection of cells of B. fragilis in blood samples even when the number was as low as 2x103colony-forming units/ml. These findings suggest that PCR-dot blot hybridization targeting nanH is a useful procedure for diagnosis of septicemia caused by B. fragilis when viable cells in blood cannot be detected by the traditional culture techniques. J. Med. Invest. 48:60-65, 2001

Keywords:Bacteroides fragilis, neuraminidase, oligonucleotide probe, septicemia, PCR

INTRODUCTION
The majority of anaerobic isolates from clinical specimens belong to the genus Bacteroides (1). The species included in the "B. fragilis group" are considered to be clinically important pathogens associated with intra-abdominal infections and abscess formation in soft tissues (2). Among these species, B. fragilis is the most virulent because this species accounts for over half of the anaerobes isolated from human infections and often causes severe septicemia with a high mortality rate in compromised hosts (2, 3). Early diagnosis and treatment with appropriate antibiotics are needed for patients infected with B. fragilis, but the traditional culture methods for anaerobes are labor-intensive and time-comsuming. In addition, if the clinical samples are not immediately cultured or kept under anaerobic conditions, obligate anaerobes are often not detected in blood cultures. Various techniques, including analysis of electrophoretic patterns of dehydrogenase (4), bacteriophage typing (5), analysis of cellular sugar and lipid compositions (6-8) and serology (9-11), have been used for rapid detection and discrimination of this anaerobe. However, all of these techniques require viable cells and many troublesome steps, and none of them have sufficient specificity and sensitivity to be used for clinical specimens.
Recently, molecular biology-based techniques have been shown to be useful for the rapid identification of many pathogenic microoganisms (12, 13). The polymerase chain reaction (PCR) targeting a specific gene is the most widely used technique in diagnostic laboratories because it is quick and it is suitable for the handling of a large number of specimens (14). Furthermore, PCR amplification enables the detection of pathogens even in culture-negative clinical specimens, since this procedure does not necessarily require viable cells (15). However, PCR amplification sometimes produces false-positive results if other bacteria have sequences similar to those of the designed PCR-primers. In a previous study, we synthesized two primer sets, F1-R1 (outer primer set) and F2-R2 (inner primer set), and used them in nested PCR to amplify the neuraminidase-encoding gene nanH of B. fragilis(16). Although these primer sets specifically amplified a part of the nanH gene of B. fragilis, one of the Bacteroides species, B. vulgatus, which possesses high neuraminidase activity, also produced a single band identical to that of B. fragilis in size when the primer set F1-R1 was used, and this false-positive band could not be excluded by electrophoresis alone. In such a case, hybridization tests with probes specific for the target are usually required to confirm the specificity of the PCR amplifications.
In the present study, we developed a digoxigenin-labeled oligonucleotide probe (named NH1), which was specific for nanH of B. fragilis, and we used it in combination with nanH-PCR for a hybridization assay. We could specifically detect B. fragilis in blood samples when PCR of a part of the gene nanH and dot-blot hybridization using NH1 were applied to model mice with bacteremia induced by challenge of this anaerobe.

MATERIALS AND METHODS
Bacterial Strains
Sixty strains of B. fragilis, including two reference strains (ATCC25285 and NCTC9343), were used in this study. The strains of other species used were B. distasonis ATCC8503, B. eggerthii ATCC27754, B. ovatus ATCC8483, B. thetaiotaomicron ATCC29148, B. uniformis ATCC8492, B. vulgatus ATCC8482, Porphyromonas asaccharolytica ATCC25260, P. endodontalis ATCC35406, P. gingivalis381 and Prevotella corporis JCM8529. All strains were cultured in GAM broth (Nissui Pharmaceutical Co., Tokyo, Japan) at 37°C under anaerobic conditions.

Preparation of Bacterial Cells for PCR
A late log-phase culture (1 ml) of each strain was centrifuged, washed with 1 ml of phosphate-buffered saline, and resuspended in0.1ml of distilled water. Each suspension was lysed by heating at100°C for10min, and10μl of the lysed preparation was used for PCR amplification of the nanH gene. PCR amplification of the nanH gene was performed as described previously (16).

Design of an Oligonucleotide Probe
We compared the nucleotide sequences of the nanH structural gene of B. fragilis strains YCH46and TAL2480. The oligonucleotide probe NH1 was synthesized on the basis of the common sequence found within F2-and R2-primer annealing sites. NH1 was labeled with digoxigenin using a DIG-labeling Kit (Boehringer GmbH, Mannheim, Germany) according to the manufacturer's instructions. The nucleotide sequence of NH1 was 5'-ATCACTATGAGTGACGGTACTTTGGTATTCCC-3'.

Dot Blot Hybridization
After PCR amplification of the nanH gene, each reaction mixture was heated at95°C for5min and chilled on an ice bath. Then, 5μl of each amplification mixture was spotted on a nylon membrane and UV-fixed. Southern hybridization was performed as described by Sambrook et al. (17). The hybridization with digoxigenin-labeled NH1 was perfomed in10ml of rapid hybridization buffer (Amersham Co., Ltd.) at 54°C for 1 hour. Post-hybridization washes were performed twice at 54°C with each washing buffer, 2xSSC (1xSSC being 0.15mM NaCl plus 15mM sodium citrate)-0.1%SDS and 0.1xSSC-0.1% SDS, respectively. The hybridization signals were detected according to the manufacturer's instructions using an alkaline phosphatase-labeled anti-digoxigenin antibody.

Preparation of Blood Samples
Three C57BL/6J mice were intraperitoneally injected with viable cells of B. fragilis. Blood samples (0.2ml) were collected by cardiac puncture at 1, 3 and 6 hours after injection, and 0.1ml of each sample was incubated anaerobically on GAM agar plates. The remainder of the samples were centrifuged, washed with 1ml of 10mM Tris-HCl and 1mM EDTA (pH8.0), and resuspended in 0.1ml of lysis buffer (10mM Tris-HCl, pH8.3, 50mM KCl, 1.5mM MgCl2 and 0.1mg of proteinase K per ml). Each suspension was incubated at 65°C for 1hour, boiled in water for 10 min, and chilled on an ice bath. Ten microliters of each solution was used as a template for PCR amplification of the nanH gene.

RESULTS AND DISCUSSION
First, we compared the nucleotide sequences of the nanH gene of B. fragilis strain YCH46 (18) with strain TAL2480 (19) to design an oligonucleotide probe that was specific for the nanH gene of B. fragilis. The nucleotide sequences of nanH from both strains were highly conserved and showed91.1% identity (Fig.1). In the present study, we chose the region at 1057-1088 (numbering in the YCH46 nanH gene) for a B. fragilis-specific probe because (i) this region of more than 30nucleotides in length is common to both strains, (ii) the guanine plus cytosine content was relatively high (43.8%),and (iii) this region corresponded to the middle of the F2-R2 annealing sites. Since a sufficient amount of the oligonucleotide probe was easily synthesized using a DNA synthesizer and non-radioisotopic labeling is required for use in diagnostic laboratories, we synthesized a digoxigenin-labeled oligonucleotide probe, NH1, based on this region. The nucleotide sequence of NH1 is described in Materials and Methods.
Dot blot hybridization using NH1 was performed against nanH PCR products from sixty strains of B. fragilis and ten other strains of related species. As shown in Fig. 2, positive spots were obtained from all strains of B. fragilis tested except for strain KMS2 (spotted at f-4.). However, strain KMS2,which had initially been identified as B. fragilis, was reidentified as B. vulgatus when we tested this strain using an API20A system (bioMerieux). In addition, none of the products from other species gave positive spots. Based on these findings, it was suggested that (i) the digoxigenin-labeled oligonucleotide probe, NH1, was specific for nanH of B. fragilis and (ii) PCR-dot blot hybridization targeting nanH was useful not only for the identification of B. fragilis but also for that of B. vulgatus (PCR with F1-R1 was positive, but dot blot hybridization with NH1 was negative.).
To assess the usefulness of nanH-PCR and the dot blot hybridization assay in clinical specimens, B. fragilis bacteremia model mice were constructed. Three 8-week-old male C57BL/6J mice were intraperitoneally injected with B. fragilis strain YCH46, and 0.2ml blood samples were collected by cardiac puncture at 1, 3 and 6hours after injection. Table1shows the results of the blood culture. Rapid translocation of B. fragilis cells from the peritoneal cavity to the blood stream was observed. This finding might represent the pathogenic potential of this species, but almost all B. fragilis cells appeared to be cleared from the blood stream within 24hours in healthy mice.
Nested PCR of the nanH gene was performed using each blood sample. Fig. 3 shows the findings of the PCR-dot blot hybridization assay using blood samples from mouse A in Table1. The 518bp bands of expected size were detected in the samples at 1, 3 and 6hours (Fig. 3A), while no band was found in the 0time control. We confirmed that these were B. fragilis-specific amplification products by dot blot hybridization with digoxigenin-labeled NH1 (Fig. 3B). These findings suggested that PCR-dot blot hybridization based on nanH enables detection of B. fragilis cells in clinical specimens with a cell number as low as 2x103cfu/ml, even if the sample tested contains non-viable cells. As shown in Fig. 3, the intensity of the PCR bands was not in proportion to the strength of the hybridization signals. The reason for this might be that the excess amount of template produced a large amount of intermediate amplification products in the early cycles of amplification and reduced the amount of specific bands, or that NH1 directly reacted with genomic DNA contained in blood samples. The latter case would mean that NH1would enable direct detection of B. fragilis cells in blood samples if there was a sufficient number of cells in the sample. It was suggested that the PCR-dot blot hybridization assay using nanH was useful for the detection and quantification of B. fragilis present in clinical specimens. Of course, NH1 can be applied for the rapid identification of cultured B. fragilis, but there are many cases in which the culture of blood from a patient is negative even when septicemia is suspected from clinical signs due to the administration of antibiotics or, particularly in cases of anaerobic infection, due to storage of anaerobes under inappropriate conditions. Furthermore, in almost all cases, clinical samples do not contain a sufficient number of cells to enable detection using direct hybridization assay. Therefore, an amplification step is needed before performing the hybridization assay to obtain accurate results.
In the present study, we demonstrated the usefulness of PCR-dot blot hybridization based on the neuraminidase-encoding gene for specific detection of B. fragilis cells using bateremia model mice. It was suggested that a combination of the method used in the present study with a traditional culture method would be useful for a clinical survey of the accurate incidence of B. fragilis infection and for differential diagnosis in patients with fever of unknown origin. However, it is necessary to determine whether this procedure is useful in the clinical setting by performing tests using various clinical samples such as pus, sputum and drainage fluid in a future study.

ACKNOWLEDGMENTS
The authors thank Drs F. Yoshimura, I. Nakamura, K. Suzuki and K. Tanaka for providing bacterial strains. The work was supported in part by a Grant-in-Aid for Developmental Scientific Research (B) (No.06557020) from the Ministry of Education, Science, Sports and Culture of Japan, and by the Kurozumi Medical Foundation.

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Received for publication August 1, 2000;accepted September ‚Q‚U, 2000.

Address correspondence and reprint requests to Yoshinari Ohnishi, M.D., PhD., Department of Bacteriology, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503,Japan and Fax:+81-886-33-7069.