Sequencing of 16S rRNA Gene: A Rapid Tool for Identification of Bacillus anthracis

Claudio T. Sacchi, Anne M. Whitney, Leonard W. Mayer, Roger Morey, Arnold Steigerwalt, Arijana Boras, Robin S. Weyant, Tanja Popovic

Disclosures

Emerging Infectious Diseases. 2002;8(10) 

In This Article

Materials and Methods

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]

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 µL) or small tissue specimens (<5 mm3) 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.

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 (https://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 (https://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 µL of 10 mM Tris, pH 8.0. The suspension was put in a Millipore 0.22-µm filter unit (Millipore, Bedford, MA), heated at 95°C for 20 min, centrifuged at 8,000 rpm for 2 min, and then used for PCR. Each final PCR reaction (100 µL) contained 5 U of Expand DNA polymerase (Roche, Mannheim, Germany); 2 µL of whole cell suspension or DNA; 10 mM Tris-HCl (pH 8.0); 50 mM KCl; 1.5 mM MgCl2; 200 µM (each) dATP, dCTP, dGTP, and dTTP; and 0.4 µM of each primer. Reactions were first incubated for 5 min at 95°C. Then 35 cycles were performed as follows: 15 s at 94°C, 15 s at the annealing temperature of 52°C, and 1 min 30 s at 72°C. Reactions were then incubated at 72°C for another 5 min. The annealing temperature for the nested PCR was 50°C. PCR products were purified with Qiaquick PCR purification kit (Qiagen).

The amplified products of approximately 1,686 bp (1,656 bp for nested PCR) were sequenced by using a modification of 16 primers as described ( Table 2 ).[11] Sequencing was performed by using a Big Dye terminator cycle sequencing kit (Applied BioSystems, Foster City, CA). Sequencing products were purified by using Centri-Sep spin columns (Princeton Separations, Adelphia, NJ) and were resolved on an Applied BioSystems model 3100 automated DNA sequencing system (Applied BioSystems). The length of sequences obtained differed for each primer but were sufficient to provide 5- to 8-fold sequence coverage. An inner fragment of 1,554 bp was obtained and analyzed by using the GCG (Wisconsin) Package, v. 10.1, (Genetics Computer Group, Madison, WI). A number was assigned for each allele of 16S rRNA gene sequence in order of elucidation; a single base change or a mixed base (more than one nucleotide determined at a single position) was considered a new 16S type. When a novel 16S type, mixed base pairs, or any discrepancies in the alignment were obtained, the 16S rRNA gene amplification and sequencing of the entire gene or parts containing the problematic region were repeated.

Sixty 16S rRNA gene sequences of B. anthracis, B. cereus, and B. thuringiensis were available in GenBank. Thirty-nine of these sequences were incomplete, contained a large number of undetermined nucleotides, or were not associated with a specific strain identification, and therefore were not used in this study. The remaining 21 sequences were identified as eight B. anthracis (AF155950 [Ames]), (AF155951 [Delta Ames]), (AF176321 [Sterne]), (AF290552 [Sterne]), (AF290553 [Vollum]), (AF155950 [Ames]), (AF155951 [Delta Ames]), and (AF176321 [Sterne]); eight B. cereus (AF155952, AF155958, AF176322, AF290546, AF290547, AF290548, AF290550, and AF290555); three B. thuringiensis (AF155954, AF155955, and AF290549); and two B. mycoides (AF155956 and AF155957). A total of 114 16S rRNA gene sequences were determined in this study (107 from isolates [GenBank accession nos. in Table 1 ] and 7 from clinical specimens [GenBank accession nos. AY138359 to AY138365).

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