One Step Multiplex PCR for Identifications at Subspecies Level of Fusobacterium nucleatum and Fusobacterium necrophorum ()
1. Introduction
Although the genus Fusobacterium was already described in 1922, a lot of new species have been found recently. This microorganism has traditionally included a variety of gram-negative bacilli that either had pointed or fusiform ends or that produced major amounts of butyric acid as an end product of metabolism [1] [2]. At present, the genus Fusobacterium comprises 33 species and 7 subspecies (http://www.bacterio.net/fusobacterium.html). This microorganism is gram-negative, non-spore forming, non-motile, pleomorphic, and obligate anaerobic bacilli, which inhabit the human oral cavity, oropharynx, upper respiratory, gastrointestinal, and female genitourinary tracts as part of the normal flora [3]. Nine species including Fusobacterium nucleatum, Fusobacterium necrophorum, Fusobacterium ulcerans, Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium necrogenes, Fusobacterium russii, and Fusobacterium varium cause human infections of the head and neck, chest, lung, liver, and abdomen [4]. The detection of Fusobacterium species in clinical specimens is important, as it may affect the prognosis and patient management. However, the identification by conventional biochemical methods can be difficult, since there are no characteristic biochemical properties to distinguish each subspecies.
F. nucleatum is the species most frequently isolated from humans. This microorganism is an opportunistic pathogen involved in various forms of periodontal diseases, extraoral infections, and colorectal cancer [5] [6], and is a highly heterogeneous species. Currently, F. nucleatum is classified into five subspecies, i.e., subsp. nucleatum, subsp. polymorphum, subsp. vincentii, subsp. fusiforme and subsp. animalis. Five subspecies have been described on the basis of electrophoretic patterns of whole-cell proteins, in addition to DNA homology, and on the basis of the electrophoretic mobility of two enzymes and DNA homology. Various other techniques have been used to confirm or challenge the validity of the type strains. Morris et al. [7] grouped these subspecies by electrophoresis of 21 alloenzymes and found overlapping identifications and entirely different groupings. On the other hand, an arbitrarily primed PCR (AP-PCR) technique, which uses two primers, has demonstrated unique profiles for five subspecies [8]. Conrads et al. [9] examined the 16S-23S internal transcribed spacer regions (ITS) sequences of all currently defined Fusobacterium spp., along with several related taxons, to determine the inter- and intraspecies and subspecies relationships. Five subspecies of F. nucleatum were differentiated both from each other and as a category from the other closely related species. Different subspecies may vary in pathogenesis relating to different levels of disease activity [10] [11] [12]. F. nucleatum subsp. nucleatum is almost isolated from periodontal disease sites, whereas F. nucleatum subsp. fusiforme and vincentii are often isolated from healthy sites as normal flora [13]. F. nucleatum subsp. animalis and polymorphum are associated with pregnancy complications [11], and F. nucleatum subsp. animalis is also associated with inflammatory bowel disease [14]. Until now, molecular technologies have been the most effective and widely accepted tools for subspecies identification [15] [16] [17]. At the subspecies level of F. nucleatum, the sequence divergences of 16S rRNA genes were only 0.6% to 1.9%, so full-length sequencing of 16S rRNA was desirable [16].
F. necrophorum is a normal inhabitant of the gastrointestinal, respiratory and genitourinary tracts of animals and humans [18] [19], and is associated with a variety of necrotic infections [20] [21] [22]. In humans, F. necrophorum primarily causes acute pharyngitis, thrombophlebitis and abscessation of the internal jugular vein, termed Lemierre’s syndrome [18] [21] [23] [24]. F. necrophorum has two major subspecies, i.e., subsp. necrophorum and subsp. funduliforme [25].
At present, F. nucleatum and F. necrophorum can be identified at the subspecies level by the sequence analysis of several target genes [26] [27], matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) [28], an AP-PCR method [8], and a PCR based typing assay [29]. However, because these methods are laborious, expensive, and time-consuming for use in the detection or discrimination of the clinical isolates of F. nucleatum and F. necrophorum at the subspecies level, epidemiological studies on the relationship between the subspecies of these microorganisms and various diseases are limited. Thus, a simple and more reliable assay for identifying F. nucleatum and F. necrophorum at the subspecies level is required. The purpose of the present study was to design primers to identify two medically important Fusobacterium species, i.e., F. nucleatum and F. necrophorum, at the subspecies level, using one-step multiplex PCR.
2. Materials and Methods
2.1. Bacterial Strains and Culture Conditions
All bacterial strains were obtained from Japan Collection of Microorganisms (JCM; Japan). The following bacterial strains were used in the present study:F. nucleatum subsp. vincentii JCM 11023, F. nucleatum subsp. nucleatum JCM 8532, F. nucleatum subsp. polymorphum JCM 12990, F. nucleatum subsp. animalis JCM 1612, F. nucleatum subsp. fusiforme JCM 11024, F. necrophorum subsp. funduliforme JCM 3724, F. necrophorum subsp. necrophorum JCM 3718, Fusobacterium equinum JCM 11174, Fusobacterium periodonticum JCM 12991, Fusobacterium simiae JCM 17465, and Fusobacterium canifelinum JCM 17464. These strains were maintained by cultivating them on Anaerobic Blood Agar (CDC), that consists of a Tryptic soy agar (Becton, Dickinson and Co., Sparks, MD, USA) supplemented with vitamin K1, hemin, L-cysteine, yeast extract, and sheep blood. These organisms were cultured at 37˚C for 48 h in an anaerobic jar with a gas pack system (AnaeroPackÒ, Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan).
2.2. Design of Species-Specific Primers for Seven Subspecies
Design of species-specific primers for five F. nucleatum subspecies and two F. necrophorum subspecies was performed as described previously [30]. Briefly, the 16S rRNA gene sequences of F. nucleatum subsp. animalis (accession no. X55404), F. nucleatum subsp. fusiforme(X55403), F. nucleatum subsp. nucleatum (AB588016), F. nucleatum subsp. polymorphum (AF287812) and F. nucleatum subsp. vincentii (AJ006964), the DNA gyrase subunit B (gyrB) gene sequences of F. nucleatum subsp. animalis (accession no. HQ008306), F. nucleatum subsp. fusiforme(HQ008304), F. nucleatum subsp. nucleatum (HQ008294), F. nucleatum subsp. polymorphum (HQ008303), F. nucleatum subsp. vincentii (HQ008296), F. necrophorum subsp. necrophorum (AY370662) and F. necrophorum subsp. funduliforme (AY370667), and the RNA polymerase B (rpoB) gene sequences of F. nucleatum subsp. animalis (accession no. GQ274961), F. nucleatum subsp. fusiforme(GQ274960), F. nucleatum subsp. nucleatum (GQ274958), F. nucleatum subsp. polymorphum (GQ274957) and F. nucleatum subsp. vincentii (GQ274959) were obtained from the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/services.html, Mishima, Japan), and a multiple sequence alignment analysis was performed with the CLUSTAL W program; i.e., the 16S rRNA gene sequences of five F. nucleatum subspecies, the gyrB gene sequences of five F. nucleatum subspecies and two F. necrophorum subspecies,and the rpoB gene sequences of e five F. nucleatum subspecies were aligned and analyzed. Homology among the primers selected for eachF. nucleatum and F. necrophorum subspecies and their respective 16S rRNA, gyrB and rpoB gene sequences was confirmed by a BLAST search.
2.3. Development of a One-Step Multiplex PCR Method Using Designed Primers
Bacterial cells were cultured in BHI supplemented with 0.5% yeast extract for 24 h, and 1 ml of the samples were then collected in microcentrifuge tubes and resuspended at a density of 1.0 McFarland standard (approximately 107 colony-forming units (CFU)/ml) in 1 ml of sterile distilled water. A total of 3.6 μl of the suspension was then used as a PCR template. The detection limit of PCR was assessed by serially diluting known numbers of bacterial cells in sterile distilled water and then subjecting each suspension to PCR. The multiplex PCR mixture contained 0.2 μM of each primer, 10 μl of 2 × MightyAmp Buffer Ver.3 (Takara Bio Inc., Shiga, Japan), 0.4 μl of MightyAmp DNA Polymerase (Takara), and 5 μl of the template in a final volume of 20 μl. PCR reactions were performed in a DNA thermal cycler (Applied Biosystems 2720 Thermal Cycler; Applied Biosystems, CA, USA). PCR conditions included an initial denaturation step at 98˚C for 2 min, followed by 30 cycles consisting of 98˚C for 10 s and 68˚C for 1 min. PCR products were analyzed by 2.0% agarose gel electrophoresis before being visualized by electrophoresis in 1 × Tris-borate-EDTA on a 2% agarose gel stained with ethidium bromide. A 100-bp DNA ladder (Takara Biomed, Shiga, Japan) was used as a molecular size marker. All experiments were performed in triplicate.
3. Results
3.1. Primer Design
Fourteen specific primers covering the upstream regions of the 16S rRNA, gyrB and rpoB gene sequences of five F. nucleatum subspecies and two F. necrophorum subspecies were designed in the present study (Figures 1-4). The specific forward primers were designated as FNAF for F. nucleatum subsp. animalis, FNFF for F. nucleatum subsp. fusiforme, FNNF for F. nucleatum subsp. nucleatum, FNPF for F. nucleatum subsp. polymorphum, FNVF for F. nucleatum subsp. vincentii, FNecNF for F. necrophorum subsp. necrophorum, and FNecFF for F. necrophorum subsp. funduliforme, whereas the specific reverse primers were designated as FNAR for F. nucleatum subsp. animalis, FNFR for F. nucleatum subsp. fusiforme, FNNR for F. nucleatum subsp. nucleatum, FNPR for F. nucleatum subsp. polymorphum, FNVR for F. nucleatum subsp. vincentii, FNecNR for F. necrophorum subsp. necrophorum, and FNecFF for F. necrophorum subsp. funduliforme. The amplicon sizes of F. nucleatum subsp. polymorphum, F. nucleatum subsp. animalis, F. nucleatum subsp. nucleatum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, F. necrophorum subsp. necrophorum, and F. necrophorum subsp. funduliforme were 101 bp, 260 bp, 535 bp, 756 bp, 912 bp, 164 bp, and 451 bp, respectively.
3.2. Multiplex PCR
3.2.1. Detection Limit
Our multiplex PCR method for identifying and detecting five F. nucleatum subspecies and two F. necrophorum subspecies, i.e., F. nucleatum subsp. animalis, F. nucleatum subsp. fusiforme, F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. vincentii, F. necrophorum subsp. necrophorum and F. necrophorum subsp. funduliforme successfully amplified DNA fragments of the expected size for each species (Figure 5 and Figure 6). The detection limit was assessed in the presence of titrated bacterial cells, and
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Figure 1. Locations and sequences of species-specific primers for the gyrB gene of F. nucleatum subsp. polymorphum. The nucleotide sequence of each primer has been underline.
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Figure 2. Locations and sequences of species-specific primers for the rpoB gene of F. nucleatum subsp. animalis, subsp. nucleatum and subsp. vincentii. The nucleotide sequence of each primer has been underline.
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Figure 3. Locations and sequences of species-specific primers for the 16S rDNA of F. nucleatum subsp. fusiforme. The nucleotide sequence of each primer has been underline.
the sensitivity of the PCR assay was between 5 × 1 and 5 × 10 CFU per PCR template (5.0 μl) for the F. nucleatum subsp. vincentii-specific primer set with strain JCM JCM 11023, the F. nucleatum subsp. nucleatum-specific primer set with strain JCM 8532, the F. nucleatum subsp. polymorphum-specific primer set
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Figure 4. Locations and sequences of species-specific primers for the gyrB gene of F. necrophorum subsp. necrophorum and subsp. funduliforme. The nucleotide sequence of each primer has been underline.
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Figure 5. Specificity of the multiplex PCR assays for F. nucleatum subspecies. The primer mixture contained FNPF, FNPR, FNAF, FNAR, FNNF, FNNR, FMFF, FNFR, FNVF and FNVR. Lanes: 1, F. nucleatum subsp. polymorphum JCM 12990; 2, F. nucleatum subsp. animalis JCM 11025; 3, F. nucleatum subsp. nucleatum JCM 8532; 4, F. nucleatum subsp. fusiforme JCM 11024; 5, F. nucleatum subsp. vincentii JCM 11023; 6, Mixture of F. nucleatum subsp. polymorphum, subsp. animalis, subsp. nucleatum, subsp. fusiforme and subsp. vincentii; 7, F. necrophorum subsp. necrophorum JCM 3718; 8, F. necrophorum subsp. fundliforme JCM 3717; 9, F. equinum JCM 11174; 10, F. periodonticum JMC 12001; 11, F. simiae JCM 17465; 12, F. canifelinum JCM 17464. M, molecular size marker (100-bp DNA ladder).
with strain JCM 12990, the F. nucleatum subsp. animalis-specific primer set with strain JCM 1612, the F. nucleatum subsp. fusiforme-specific primer set with strain JCM 11024, the F. necrophorum subsp. funduliforme-specific primer set with strain JCM 3724 and the F. necrophorum subsp. necrophorum-specific primer set with strain JCM 3718 (data not shown).
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Figure 6. Specificity of the multiplex PCR assays for F. necrophorum subspecies. The primer mixture contained FNecNF, FNecNR, FNecFF and FNecFR. Lanes: 1, F. necrophorum subsp. necrophorum JCM 3718; 2, F. necrophorum subsp. fundliforme JCM 3717; 3, Mixture of F. necrophorum subsp. necrophorum and subsp. fundliforme; 4, F. nucleatum subsp. polymorphum JCM 12990; 5, F. nucleatum subsp. animalis JCM 11025; 6, F. nucleatum subsp. nucleatum JCM 8532; 7, F. nucleatum subsp. fusiforme JCM 11024; 8, F. nucleatum subsp. vincentii JCM 11023; 9, F. equinum JCM 11174; 10, F. periodonticum JMC 12001; 11, F. simiae JCM 17465; 12, F. canifelinum JCM 17464. M, molecular size marker (100-bp DNA ladder).
3.2.2. Assay of Representative Candida Species Other than Eight Medically Important Species
As representativeFusobacterium species other than F. nucleatum and F. necrophorum subspecies targeted in this study, Fusobacterium equinum, Fusobacterium periodonticum, Fusobacterium simiae and Fusobacterium canifelinum were subjected to PCR using the designed primer sets. No amplicons were produced from any of representativeFusobacterium species other than F. nucleatum and F. necrophorum (Figure 5 and Figure 6).
4. Discussion
To develop a PCR-based technique more applicable for clinical use than conventional PCR, we established a one-step multiplex PCR system for identifying and detecting simultaneously two medically important Fusobacterium species, using only two PCR tube per sample. A multiplex-PCR method is a rapid tool that allows for the simultaneous amplification of more than one sequence of target DNA in a single reaction, thereby saving time and reagents [31]. The most significant problem with regard to this method is the possibility of hybridization among the different sequences of primers. Bi et al. reported a PCR strategy allowing the identification at the subspecies level of F. nucleatum as same as the present study [29]. This PCR method was based on the amplification of the fragments from the subspecies marker genes in a single PCR. However, because this method was not able to identify F. nucleatum subsp. fusiforme and adopted a conventional PCR, it was not suitable for routine clinical laboratory identification ofFusobacterium species.
The phylogenetic differences are not only expressed in the sequence information itself but also by the different lengths of amplificons and, in some cases, in the formation of distinct band patterns (in gel electrophoresis) resulting from variations among the rrn operons in the same strain [32] [33]. The taxonomy of fusobacterial species and some related genera and species is still a scientific riddle, especially with respect to the five controversial subspecies in Fusobacterium nucleatum [34] and the two in Fusobacterium necrophorum [35]. In the present study, we designed species-specific primers with the already mentioned means, for the identification at the subspecies level of two medically important Fusobacterium species, i.e., F. nucleatum and F. necrophorum, with a PCR method. These primers were able to distinguish each Fusobacterium species at the subspecies level and did not display cross-reactivity with representative Fusobacterium species other than the two species targeted in this study. Moreover, we developed a one-step multiplex PCR method with the ability to identify and differentiate two medically important Fusobacterium species at the subspecies level using only each one PCR tubes per sample. Species-specific primers for seven subspecies were designed based on the sequences of 16S rRNA, gyrB and rpoB gene. The protein-coding genes that have been tested for the assessment of microbial diversity include the genes for DNA gyrase subunit B (gyrB) [36], RNA polymerase subunit B (rpoB) [37], the TU elongation factor (tuf) [38], the 60 kDa chaperonin protein (cpn60) [39]. The gyrB and rpoB genes have a number of potential advantages. Previous reports have shown these markers to be suitable for phylogenetic analyses, as it provides a better resolution at species level than the 16S rRNA gene [40].
Our multiplex PCR method is easy because the use of MightyAmp DNA Polymerase Ver.3 (Takara) means that DNA extraction may be avoided, and the subspecies identification and detection using this method only takes approximately 2 hours. Thus, the method described herein will allow the prevalence at the subspecies level of F. nucleatum and F. necrophorum and their involvement in the various infections, to be fully clarified in future studies.
5. Conclusion
Our developed one-step multiplex PCR method is accurate, specific, cost-effective, time-saving, and worked without requiring DNA extraction. It is predicted that our multiplex PCR method can be used in routine practice in clinical microbiology laboratories to identify F. nucleatum and F. necrophorum at the subspecies level.
Authors and Affiliations
Umezawa K, Hayashi S, Tsuzukibashi O, Fukatsu A, Fuchigami M, Uchibori S, Komine C and Asano T corrected the data. Umezawa K, Tsuzukibashi O, Wakami M, Kobayashi T and Fukumoto M drafted and wrote the manuscript. The concept of this manuscript was devised by Umezawa K. All authors read and approved the final manuscript.