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J Clin Microbiol. Sep 2008; 46(9): 3139–3143.
Published online Jul 23, 2008. doi:  10.1128/JCM.00275-08
PMCID: PMC2546735

Molecular Method for Discrimination between Francisella tularensis and Francisella-Like Endosymbionts[down-pointing small open triangle]

Abstract

Environmental studies on the distribution of Francisella spp. are hampered by the frequency of Francisella-like endosymbionts that can produce a misleading positive result. A new, efficient molecular method for detection of Francisella tularensis and its discrimination from Francisella-like endosymbionts, as well as two variants associated with human disease (unusual F. tularensis strain FnSp1 and F. tularensis subsp. novicida-like strain 3523), is described. The method is highly specific and sensitive, detecting up to one plasmid copy or 10 genome equivalents.

Francisella spp., which are gram-negative cocobacilli, are the etiological agents of tularemia. Currently, there are four subspecies recognized within the F. tularensis species (F. tularensis subsp. tularensis, holarctica, mediasiatica, and novicida), which present differences in virulence and geographical distribution (16). Recently, there have also been descriptions of strains associated with human diseases that presented atypical features with uncertain taxonomic position (6, 11, 19). Based on 16S rRNA gene sequences, several other organisms have been classified as probable members of the Francisellaceae family, including Wolbachia persica and “Francisella-like endosymbionts” (FLEs) (13, 15, 17). Although FLEs are believed to be nonpathogenic to humans, their pathogenicity to guinea pigs and hamsters has been demonstrated (4), and they have been detected recently in free-living small mammals (A. Toledo, R. Escudero, A. S. Olmeda, J. F. Barandika, M. A. Casado-Nistal, I. Jado, H. Gil, A. L. García-Pérez, and P. Anda, submitted for publication). The homology found in the 16S rRNA between Francisella spp. and FLEs poses a potential problem for laboratories that screen environmental samples such as ticks, due to PCR cross-reactivity (12). The significance of this problem has been heightened by the classification of F. tularensis as a category A agent of bioterrorism (5). The homology between the sequence of the lipoprotein-coding lpnA gene of Francisella pathogens and that of FLEs is high, although there is variability, and FLEs conform to a phylogenetic clade separate from F. tularensis species (15). We selected a 233-bp fragment on a variable region of lpnA that is able to differentiate between F. tularensis pathogens and FLEs by amplification and further hybridization with specific probes by reverse line blotting (RLB).

Primers and probes were designed with Oligo6 software (Molecular Biology Insights, Inc., West Cascade, CO), and the Basic Local Alignment Search Tool (BLASTn) was used for a preliminary assessment of the oligonucleotides' specificity (1). Available sequences from GenBank were aligned and analyzed by ClustalX and Mega4 software (8, 18). Figure Figure11 shows the alignment of representative sequences.

FIG. 1.
Sequence alignment of a 233-nucleotide fragment of lpnA of representative subspecies of F. tularensis and FLE, as well as F. philomiragia (nucleotide positions 593 to 825, as in F. tularensis subsp. holarctica strain LVS [GenBank accession no. ...

The region covering nucleotide positions 593 to 825 of lpnA (as in F. tularensis subsp. holarctica strain LVS; GenBank accession no. M32059) was amplified from each sample by using degenerated primers (Table (Table1)1) that allowed the amplification of DNA from all of the known subspecies and variants of F. tularensis and all of the described FLEs (2). Also, an internal amplification control was added to evaluate the presence of PCR inhibitors as described previously (10). PCR was performed in a 50-μl reaction volume with 10 mM Tris-HCl, 50 mM KCl, 3 mM MgCl2, 200 μM of each deoxynucleoside triphosphate (dNTP) (Promega, Madison, WI), and 1.5 U of Taq gold DNA polymerase (Applied Biosystems, Branchburg, NJ). Primers were used at a final concentration of 0.5 μM. PCR cycling included an initial denaturing step of 9 min at 94°C, followed by 40 cycles of 15 s at 94°C, 1 min at 60°C, and 4 min at 65°C and a final elongation step of 7 min at 65°C. The overall time required for PCR was 3 h.

TABLE 1.
Probes and primers used in the study

For the hybridization, a Biometra OV3 mini hybridization oven (Cultek, S.L., Madrid, Spain) was used, and the RLB was performed as previously described (10), with few modifications as follows: 12.8 pmol/μl of each probe (Table (Table1)1) was attached to the membrane, the selected temperature for hybridization was 48°C, and the subsequent incubation with streptavidin-peroxidase conjugate (Roche Farma, S.S., Madrid, Spain) and the washing steps were performed at 40°C. Super Signal West Dura Extended Duration Substrate (Pierce Biotechnology, Rockford, IL) was used for the development of the membrane. The overall time required for the RLB was 3.5 h.

Recently, a study published by Nübel et al. (14) showed different lpnA sequences within F. tularensis subspecies. This difference consists of a deletion of 36 bp in the region between nucleotide positions 662 and 697 (as in F. tularensis subsp. holarctica strain LVS; GenBank accession no. M32059). Consequently, the following probes were designed (Table (Table1):1): a generic probe (P-FRAG) that hybridized with the different subspecies of F. tularensis, FLEs and other variants described (FnSp1 and 3523); a probe for all the F. tularensis subspecies which do not present that deletion (P-TUL); a generic probe (P-ENDO2) for all known FLEs; and an additional probe (P-FnSp1) for two atypical human isolates (FnSp1 and 3523). It is remarkable that the probe P-TUL will catch all the F. tularensis subspecies that show a complete lpnA sequence. Given this, the difference in reactivities observed with P-FRAG and P-TUL will differentiate among those two sequence types.

To be used as positive controls, since the strains were not available, synthetic DNA fragments were constructed following the sequences of lpnA from an FLE from Dermacentor variabilis (GenBank accession no. AY375420) (15) and F. tularensis subsp. novicida-like isolate 3523 (GenBank accession no. AY243029) (19) and by using overlapping primers up to 75 bp long in consecutive PCRs (Table (Table2).2). The fragments were cloned in pGEM-T Easy vectors (Promega Biotech Ibérica, S.L., Madrid, Spain) by following the manufacturer's instructions and sequenced to check their fidelity. The plasmid copy numbers were quantified by spectrophotometry with a NanoDrop ND-1000 spectrophotometer (Nucliber, Madrid, Spain). Genomic DNAs from F. tularensis subsp. holarctica strain LVS, F. tularensis subsp. tularensis strain B-38, F. tularensis subsp. novicida strain Utah 112, and the atypical isolate FnSp1 (6), all obtained after purification with a QIAamp DNA minikit (IZASA S.A., Barcelona, Spain), were used as positive controls as well (Table (Table2).2). To determine the sensitivity of the assay, 1, 10, and 102 copies/genomic equivalent (GE) of each plasmid/strain were tested per reaction.

TABLE 2.
Strains and organisms included in this study

DNA from clinical and environmental sources was obtained using a QIAamp DNA blood extraction kit (Qiagen). Clinical samples from patients consisted of lymph node aspirates, skin exudates, and skin biopsy specimens from the outbreak of tularemia that occurred in Spain in 1997 (9). Questing ticks and small mammals from Madrid (Toledo et al., submitted) as well as questing ticks from the Basque Country (3) were also included in this study.

The sensitivity of the method was excellent. One plasmid copy and 10 GE were detected when synthetic or genomic DNAs of the positive controls were tested, respectively (Fig. (Fig.2A,2A, lanes 1 to 18). As expected, the strains of F. tularensis subsp. holarctica (Fig. (Fig.2A,2A, lanes 19 to 26) reacted with P-FRAG and P-TUL, and F. tularensis subsp. novicida strain Fx1 reacted with P-FRAG and P-TUL, whereas Fx2 only reacted with P-FRAG, since this strain presents the deletion mentioned above (Fig. (Fig.2A,2A, lanes 29 and 30). Finally F. philomiragia did not react with any of the designed probes (Fig. (Fig.2A,2A, lanes 27 and 28). In addition, the test was performed again with 10 GE of F. tularensis subsp. holarctica strain or 1 plasmid copy of synthetic DNA of FLEs in the presence of foreign DNA free of pathogens (300 ng of human DNA, 300 ng of DNA from Ixodes ricinus and Rhipicephalus sanguineus specimens, and 300 ng of a C3H mouse DNA), showing no loss of sensitivity (Fig. (Fig.2B,2B, lanes 14 to 21). Sources for the DNA as well as a summary of the results are shown in Table Table22.

FIG. 2.
RLB results of different samples, as in Table Table2.2. (A) Lanes 1 to 3, 102, 10, and 1 GE, respectively, F. tularensis subsp. holarctica strain LVS; lanes 4 to 6, 102, 10, and 1 GE, respectively, F. tularensis subsp. tularensis strain B-38; ...

No cross-reaction was observed either with any of the microorganisms tested for specificity (102 GE of different related pathogens or an estimated similar amount of DNA from intracellular pathogens, such as Anaplasma phagocytophilum, Borrelia burgdorferi, Coxiella burnetii, Legionella pneumophila, Mycoplasma pneumoniae, Chlamydia pneumoniae, Rickettsia conorii, and Orientia tsutsugamushi) (Fig. (Fig.2B,2B, lanes 2 to 9) or with 300 ng of host DNA or vector DNA free of pathogens (Fig. (Fig.2B,2B, lanes 10 to 13).

All of the tularemia samples from patients (23 samples) were positive by PCR-RLB, yielding a sensitivity of 100% (data not shown). A representative sample of this result is shown in Fig. Fig.2B,2B, lanes 22 to 27. FLEs were detected in ticks collected from vegetation as well as from small mammals (Fig. (Fig.2B,2B, lanes 28 to 33).

These results demonstrated that this method has shown excellent sensitivity and specificity values and also has the ability to differentiate among FLEs and known human pathogens, including atypical human isolates (FnSp1 and 3523) not included in routine practice for identification due to rare occurrence. Consequently, it can be used in epidemiological surveys, especially in environmental studies where FLEs are expected to be present. The primers used in this method are generic for any known F. tularensis strain and FLEs, and sequencing of new variants that could arise in the future will provide us with the possibility of designing new probes for their detection.

Kugeler et al. have recently described a method that differentiates between F. tularensis and FLEs (12). Their approach, using a multitarget TaqMan assay, has been shown to be both sensitive and specific, but sequencing of 16S rRNA was necessary for the identification of FLEs in the samples tested. The methodology proposed here is easy to set up and robust, as has been shown with other pathogens (7, 10). Moreover, this method has been tested under field conditions and is able to detect FLEs in both ticks and small mammals, as well as F. tularensis in ticks and humans, all without sequencing. However, considering the available information regarding FLEs, samples reactive to P-ENDO2, specific for FLEs, should be subjected to sequencing of additional genes to further assess the specificity of the probe.

As a result of increased concern over the terrorist use of agents such as F. tularensis, an extensive understanding of the diversity and distribution of this organism is required. Here, we have described a simple method for the detection and differentiation among F. tularensis subspecies, FLEs, and new atypical Francisella variants recently described as associated with human disease that has high sensitivity and specificity. The generic approach used allows the detection of all known members of these groups. Moreover, this method represents an excellent and versatile tool for performing clinical and environmental studies.

Acknowledgments

This study has been supported by grant EM03/06 from Instituto de Salud Carlos III.

We gratefully acknowledge M. Forsman (Swedish Defense Research Agency, Umea, Sweden) for providing F. tularensis strains.

Footnotes

[down-pointing small open triangle]Published ahead of print on 23 July 2008.

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