Sequencing of 16S rRNA Gene: a rapid tool for identification of Bacillus anthracis - Bioterrorism-Related Antrax

Emerging Infectious Diseases, Oct, 2002 by Claudio T. Sacchi, Anne M. Whitney, Leonard W. Mayer, Roger Morey, Arnold Steigerwalt, Arijana Boras, Robin S. Weyant, Tanja Popovic

In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis from other closely related spore-forming Bacillus species. During the recent outbreak of bioterrorism-associated anthrax, we sequenced the 16S rRNA genes from these species to evaluate the potential of 16S rRNA gene sequencing as a diagnostic tool. We found eight distinct 16S types among all 107 16S rRNA gene sequences that differed from each other at 1 to 8 positions (0.06% to 0.5%). All 86 B. anthracis had an identical 16S gene sequence, designated type 6; 16S type 10 was seen in all B. thuringiensis strains; six other 16S types were found among the 10 B. cereus strains. This report describes the first demonstration of an exclusive association of a distinct 16S sequence with B. anthracis. Consequently, we were able to rapidly identify suspected isolates and to detect the B. anthracis 16S rRNA gene directly from culture-negative clinical specimens from seven patients with laboratory-confirmed anthrax.

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The gram-positive, rod-shaped, and spore-forming bacterium Bacillus anthracis is the cause of the acute and often lethal disease anthrax. Phenotypic characteristics commonly used to differentiate B. anthracis from closely related B. cereus and B. thuringiensis, such as susceptibility to [beta]-1actam antibiotics, lack of motility, lack of hemolysis on sheep blood agar (SBA) plate, and susceptibility to T-phage lysis, may vary among different Bacillus species strains, hampering their identification and differentiation. Phenotypically and genotypically B. thuringiensis can be differentiated from B. cereus by the presence of the CRY crystal protein and plasmid-encoded cry genes (1), but if this plasmid were lost, B. thuringiensis could no longer be distinguished from B. cereus (1). The sequence of the 16S rRNA gene has been widely used as a molecular clock to estimate relationships among bacteria (phylogeny), but more recently it has also become important as a means to identify an unknown bacterium to the genus or species level. The 16S rRNA gene sequences of B. anthracis, B. cereus, and B. thuringiensis have high levels of sequence similarity (>99%) that support their close relationships shown by DNA hybridization (2-7). A limited number of 16S rRNA sequences of B. anthracis (7 sequences), B. cereus (34 sequences), and B. thuringiensis (16 sequences) have been available at GenBank. Although those sequences are of different lengths and qualities, in complementary regions they differ from each other by no more than a few nucleotides. Therefore, this minimal level of diversity seen in the 16S rRNA of B. anthracis, B. cereus, and B. thuringiensis was thought to be an obstacle for using 16S rRNA gene sequencing to identify and differentiate these three species. The bioterrorism events of October 2001 prompted us to evaluate several new molecular approaches to rapidly identify B. anthracis. We determined the entire 16S rRNA sequences in a large number of representative strains of B. anthracis, B. cereus, and B. thuringiensis to evaluate the potential of 16S rRNA sequencing not only to rapidly identify B. anthracis in culture, but also to detect B. anthracis directly in clinical specimens.

Materials and Methods

Bacterial Strains

A total of 107 strains were included in this study. Of 86 B. anthracis isolates analyzed (Table 1), 18 were selected to represent a wide range of temporal (1937-1997), geographic (16 countries), and source diversity (soil, animals, or humans). Fourteen reference and standard strains, such as the Vollum, Ames, Pasteur, New Hampshire, V770, and Sterne strains, were also included. The remaining 54 strains were isolated from October to December 2001 during the bioterrorism-associated anthrax outbreak in the United States. Ten B. cereus and 11 B. thuringiensis strains were also analyzed by 16S rRNA sequencing. All strains were identified by standard microbiologic procedures and according to the Laboratory Response Network diagnostic criteria (9,10).

Clinical Specimens

We analyzed 198 clinical specimens (76 blood, 30 tissue, 16 pleural fluid, 37 serum, 6 cerebrospinal fluid, and 33 other specimens). Sixty-nine specimens were obtained from patients with laboratory-confirmed anthrax (55 specimens from 11 inhalational cases and 14 from 7 cutaneous cases). DNA was extracted from fluid (200 [micro]L) or small tissue specimens (<5 [mm.sup.3]) according to manufacturer's instructions with a Qiagen DNA Mini Kit (Qiagen, Valencia, CA). All 198 DNA samples were analyzed for 16S rRNA gene amplification and products sequenced.

Polymerase Chain Reaction (PCR)

A 1,686-bp fragment of DNA, including the 1,554-bp 16S rRNA gene, was amplified from all 107 Bacillus species strains by using primers 67F and 1671R (Table 2). For clinical samples, we used the initial DNA amplicon as a template in a nested PCR with a second set of internal primers, 23F and 136R (Table 1). Both sets of primers were designed from the B. anthracis genome sequence (http://www.tigr.org). The full-length size of B. anthracis 16S rRNA gene (1,554 bp) was determined from an alignment of the 16S rRNA genes from Escherichia coli, Neisseria gonorrhoeae (GenBank accession nos. J01859 and X07714, respectively), and the 16S rRNA gene regions of the B. anthracis genome sequence (http:// www.tigr.org). Whole cell suspensions or DNA extracts were used for PCR of isolates or clinical samples, respectively. For whole cell suspensions, a single colony from an SBA plate was resuspended in 200 [micro]L of 10 mM Tris, pH 8.0. The suspension was put in a Millipore 0.22-[micro]m filter unit (Millipore, Bedford, MA), heated at 95[degrees]C for 20 min, centrifuged at 8,000 rpm for 2 min, and then used for PCR. Each final PCR reaction (100 [micro]L) contained 5 U of Expand DNA polymerase (Roche, Mannheim, Germany); 2 [micro]L of whole cell suspension or DNA; 10 mM Tris-HCl (pH 8.0); 50 mM KCl; 1.5 mM Mg[Cl.sub.2]; 200 [micro]M (each) dATP, dCTP, dGTP, and dTTP; and 0.4 [micro]M of each primer. Reactions were first incubated for 5 min at 95[degrees]C. Then 35 cycles were performed as follows: 15 s at 94[degrees]C, 15 s at the annealing temperature of 52[degrees]C, and 1 min 30 s at 72[degrees]C. Reactions were then incubated at 72[degrees]C for another 5 min. The annealing temperature for the nested PCR was 50[degrees]C. PCR products were purified with Qiaquick PCR purification kit (Qiagen).