Sequencing of 16S rRNA Gene: a rapid tool for identification of Bacillus anthracis. (Bioterrorism-Related Antrax).In a bioterrorism event, a tool is needed to rapidly differentiate Bacillus anthracis Bacillus anthracis Infectious disease A gram-positive organism which causes often fatal infections when its endospores–resistant to heat, drying, UV light, gamma radiation, and many disinfectants–enter the body and cause septicemia Military medicine from other closely related spore-forming Bacillus bacillus (bəsĭl`əs), any rod-shaped bacterium or, more particularly, a rod-shaped bacterium of the genus Bacillus. Some bacterium in the genus cause disease, for example B. species. During the recent outbreak of bioterrorism-associated anthrax anthrax (ăn`thrăks), acute infectious disease of animals that can be secondarily transmitted to humans. It is caused by a bacterium (Bacillus anthracis , 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 cereus: see cactus. cereus Any of various large cacti (genus Cereus and related genera) of the western U.S. and tropical New World, including the saguaro and the organ-pipe cactus (Lemairocereus thurberi, also L. marginatus or C. thurberi). 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. *********** The gram-positive, rod-shaped, and spore-forming bacterium bacterium /bac·te·ri·um/ (bak-ter´e-um) pl. bacte´ria [L.] in general, any of the unicellular prokaryotic microorganisms that commonly multiply by cell division, lack a nucleus or membrane-bound organelles, and possess a cell Bacillus anthracis is the cause of the acute and often lethal disease anthrax. Phenotypic phe·no·type n. 1. a. The observable physical or biochemical characteristics of an organism, as determined by both genetic makeup and environmental influences. b. 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 motility /mo·til·i·ty/ (mo-til´ite) the ability to move spontaneously.mo´tile Motility Motility is spontaneous movement. , lack of hemolysis hemolysis (hĭmŏl`ĭsĭs), destruction of red blood cells in the bloodstream. Although new red blood cells, or erythrocytes, are continuously created and old ones destroyed, an excessive rate of destruction sometimes occurs. on sheep blood agar blood agar n. A nutrient culture medium that is enriched with whole blood and used for the growth of certain strains of bacteria. (SBA SBA abbr. Small Business Administration Noun 1. SBA - an independent agency of the United States government that protects the interests of small businesses and ensures that they receive a fair share of government ) plate, and susceptibility to T-phage lysis lysis /ly·sis/ (li´sis) 1. destruction or decomposition, as of a cell or other substance, under influence of a specific agent. 2. mobilization of an organ by division of restraining adhesions. 3. , 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 plasmid Genetic element not contained within a chromosome. It occurs in many bacterial strains. Plasmids are circular DNA molecules that replicate independently of the bacterial chromosome. They are not essential for the bacterium but may give it a selective advantage. 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 DNA hybridization Molecular medicine A technique for determining the presence of a target DNA in a sample of tissue or cells. See HLA analysis, Paternity testing, RFLP analysis. (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 New Hampshire, one of the New England states of the NE United States. It is bordered by Massachusetts (S), Vermont, with the Connecticut R. forming the boundary (W), the Canadian province of Quebec (NW), and Maine and a short strip of the Atlantic Ocean (E). , 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 United States, officially United States of America, republic (2005 est. pop. 295,734,000), 3,539,227 sq mi (9,166,598 sq km), North America. The United States is the world's third largest country in population and the fourth largest country in area. . 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 according to prep. 1. As stated or indicated by; on the authority of: according to historians. 2. In keeping with: according to instructions. 3. the Laboratory Response Network diagnostic criteria (9,10). Clinical Specimens We analyzed 198 clinical specimens (76 blood, 30 tissue, 16 pleural fluid pleural fluid n. The thin film of serous fluid between the visceral and parietal pleurae. , 37 serum, 6 cerebrospinal fluid cerebrospinal fluid (CSF) Clear, colourless liquid that surrounds the brain and spinal cord and fills the spaces in them. It helps support the brain, acts as a lubricant, maintains pressure in the skull, and cushions shocks. , 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 cutaneous /cu·ta·ne·ous/ (ku-ta´ne-us) pertaining to the skin. cu·ta·ne·ous adj. Of, relating to, or affecting the skin. Cutaneous Pertaining to the skin. cases). DNA DNA: see nucleic acid. DNA or deoxyribonucleic acid One of two types of nucleic acid (the other is RNA); a complex organic compound found in all living cells and many viruses. It is the chemical substance of genes. 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 Gene amplification The process by which a cell specifically increases the copy number of a particular gene to a greater extent than it increases the copy number of genes composing the remainder of the genome (all the genes which make up the genetic machinery and products sequenced. Polymerase Chain Reaction polymerase chain reaction (pŏl`ĭmərās') (PCR), laboratory process in which a particular DNA segment from a mixture of DNA chains is rapidly replicated, producing a large, readily analyzed sample of a piece of DNA; the process is (PCR PCR polymerase chain reaction. PCR abbr. polymerase chain reaction 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 Escherichia coli (ĕsh'ərĭk`ēə kō`lī), common bacterium that normally inhabits the intestinal tracts of humans and animals, but can cause infection in other parts of the body, especially the urinary tract. , Neisseria gonorrhoeae Neisseria gon·or·rhoe·ae n. Gonococcus. Neisseria gonorrhoeae The bacterium that causes gonorrhea. It cannot survive for any length of time outside the human body. (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 DNA polymerase /DNA po·lym·er·ase/ (pah-lim´er-as) any of various enzymes catalyzing the template-directed incorporation of deoxyribonucleotides into a DNA chain, particularly one using a DNA template. (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 annealing (ənēl`ĭng), process in which glass, metals, and other materials are treated to render them less brittle and more workable. 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). 16S rRNA Sequence Determination 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 (1) A character that ends a string of alphanumeric characters. (2) A hardware component that is connected to the last peripheral device in a series or the last node in a network. cycle sequencing kit (Applied BioSystems Applied Biosystems, Inc. (formerly NASDAQ: ABIO) is the original name of a pioneer biotechnology company founded in 1981 in Foster City, California, among the Silicon Valley cities of the southern San Francisco Bay Area. , 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 DNA sequencing The determination of the sequence of nucleotides in a sample of DNA. 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 GCG Genetics Computer Group GCG Glucagon GCG Good Corporate Governance GCG Global Consumer Group GCG Global Church of God GCG Generalized Conjugate Gradient GCG Global Change Game GCG Geological Curators' Group GCG Giant-Cell Granuloma (Wisconsin) Package, v. 10.1, (Genetics Computer Group, Madison, WI). A number was assigned for each allele allele (əlēl`): see genetics. allele Any one of two or more alternative forms of a gene that may occur alternatively at a given site on a chromosome. of 16S rRNA gene sequence in order of elucidation e·lu·ci·date v. e·lu·ci·dat·ed, e·lu·ci·dat·ing, e·lu·ci·dates v.tr. To make clear or plain, especially by explanation; clarify. v.intr. To give an explanation that serves to clarify. ; 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. GenBank 16S rRNA Gene Sequences and Accession Numbers Accession number may mean:
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. AY 138359 to AY 138365). Results 16S rRNA Gene Sequence Diversity The 1,554-bp nucleotide sequences of the entire 16S rRNA gene from all 107 Bacillus species strains were aligned and compared. Differences were found at eight single nucleotide positions (positions 1, 2, 3, 4, 6, 9, 12, and 13), and no gaps were present. When 21 Bacillus 16S rRNA sequences from GenBank were added to the alignment, five additional positions with differences (positions 5, 7, 8, 10, and 11) were located (Table 3). The 13 positions of differences were distributed throughout the gene (Table 3). In six of these positions (positions 1, 2, 3, 4, 6, and 12), more than one nucleotide was detected (mixed nucleotides) (Table 3). These results indicated that the strain contained multiple rRNA operons with slightly different 16S rRNA gene sequences. We found eight different 16S types among the 107 16S rRNA genes from our collection of isolates (Table 3). All 86 B. anthracis had an identical sequence, 16S type 6, containing a single mixed base, a W(A/T) at position 12, not found in the other two species. 16S type 10 was seen in all 11 B. thuringiensis strains, and a single mixed base pair was identified in all strains at position 6. Six other 16S types were found among the 10 B. cereus strains. Three additional 16S types were found among the 18 GenBank sequences that we analyzed. 16S types 1, 4, and 5 correlated to B. mycoides, B. thuringiensis, and B. cereus, respectively (Table 3). Five B. anthracis sequences from GenBank were identical to the 16S type 6 found in all our 86 B. anthracis isolates, and three were identical to the 16S type 7 found in B. cereus. 16S rRNA Sequencing Directly in Clinical Specimens We detected 16S rRNA genes in 7 (3.5%) of 198 clinical samples: all were 16S type 6 characteristic for B. anthracis. None of the seven specimens were culture positive (Table 4), although all specimens had been collected from patients with laboratory-confirmed anthrax. Discussion The goal of this study was to evaluate the potential of 16S rRNA sequencing to rapidly identify B. anthracis in cultures. We found that 16S rRNA genes of B. anthracis were highly conserved; only one 16S type (16S type 6) was identified in all 86 strains tested. However, not all B. anthracis 16S rRNA genes sequences in GenBank are type 6. Three of the eight B. anthracis 16S rRNA sequences are reported as type 7, a type that, in our study, we found exclusively among the B. cereus strains. The only difference between type 7 and type 6 is a mixed base pair at position 1146. The strain designations of two of these three 16S type 7 B. anthracis strains in GenBank are Ames and Sterne. We did not acquire these particular strains from the submitting laboratory, but the one Ames and two Sterne strains (obtained from different sources) in our collection were consistently type 6. A third Sterne strain 16S rRNA sequence in GenBank is also type 6. One possible explanation for these different 16S rRNA sequencing results may be the use of different sequencing approaches, such as using cloned DNA versus genomic DNA genomic DNA n. The full complement of DNA contained in the genome of a cell or organism. as template. In sequencing clones, one allele may be missed if only a few clones are sequenced, not representing the total diversity. In this case, the position with the mixed base would not be detected. If both types 6 and 7 exist in B. anthracis, the difference may be due to recombination recombination, process of "shuffling" of genes by which new combinations can be generated. In recombination through sexual reproduction, the offspring's complete set of genes differs from that of either parent, being rather a combination of genes from both parents. , mutation, or loss of an allele. The type 7 B. anthracis sequences in GenBank are unpublished; therefore, we do not know if the genes were cloned and, if so, how many clones were sequenced. The complete B. anthracis genome was posted at http:// www.tigr.org/tigr-scripts/ufmg/ReleaseDate.pl on May 7, 2002. The genome has 11 rRNA operons. There are 10 positions in the 16S rRNA gene where the nucleotides are not identical among the 11 rRNA operons, but the DNA sequencing software scores only one of them as a mixed base 100% of the time. This position is 1146, where five 16S rRNA genes contain Ts and six have As in a 54%:46% ratio. In this case, the base-calling software (GCG; Genetics Computer Group) always assigns a W at that position. At position 1137, there are seven Gs and four As, a 64%:36% ratio, but the position is scored as a G, the predominant base. In eight positions, a 9%:91% ratio is present. For example, at position 1047 are one T and 10 Cs. In these cases, the nucleotide is called as the predominant base by the base-calling software. The quality of DNA sequences DNA sequence Genetics The precise order of bases–A,T,G,C–in a segment of DNA, gene, chromosome, or an entire genome. See Base pair, Base sequence analysis, Chromosome, Gene, Genome. generated in laboratories has been greatly improved by the introduction of automated sequencing systems and DNA alignment software, but other factors, such as the purity of the DNA template and number of overlapping nucleotide fragments in the alignment, contribute to the reliability of the final sequence. Mixed base pairs are clearly the result of sequence differences between different rRNA operons and not due to any sequencing artifacts artifacts see specimen artifacts. . In this study, the length of the fragment sequences varied for each primer, but they were of sufficient length to provide 5- to 8-fold sequence coverage in both directions. This 5-8 sequence overlap simplifies identifying and clarifying positions with double signals, increasing the confidence in our final consensus sequence. The occurrence of mixed base pairs in rRNA sequences is well known and accepted (15-19). The Ribosomal Database Project Web site shows that operon heterogeneity het·er·o·ge·ne·i·ty n. The quality or state of being heterogeneous. heterogeneity the state of being heterogeneous. has been documented in several different bacterial species (http://rrndb.cme.msu.edu/rrndb/rrn_table.pdf). In addition, we did not observe mixed base pairs in single-copy genes such as pagA and a variety of others. A previous study of a small set of Bacillus strains isolated from soil demonstrated the diversity of 16S rRNA genes of both B. cereus and B. thuringiensis (15). Our results confirm the diversity among B. cereus strains, although we did not find diversity among B. thuringiensis strains. The lack of diversity in our collection of B. thuringiensis strains may be associated with natural selection with human host; 6 of 11 of our B. thuringiensis strains were isolated from humans. Direct Amplification of 16S rRNA from Clinical Samples Even though B. anthracis is present at high levels (up to [10.sup.8]/mL) in the blood of patients with anthrax and will readily grow on standard bacteriologic bac·te·ri·ol·o·gy n. The study of bacteria, especially in relation to medicine and agriculture. bac·te media, as for other bacteria, specimens collected after the administration of antimicrobial antimicrobial /an·ti·mi·cro·bi·al/ (-mi-kro´be-al) 1. killing microorganisms or suppressing their multiplication or growth. 2. an agent with such effects. therapy may fail to grow B. anthracis. Laboratory confirmation for the two patients with inhalational anthrax whose specimens were analyzed (patient #10i [12], and patient #11i [13]) was achieved by isolation and identification of B. anthracis from clinical samples at the medical facility where the patients were treated. Generally, for all patients, isolates themselves were forwarded to the appropriate public health laboratory and then to the Centers for Disease Control and Prevention Centers for Disease Control and Prevention (CDC), agency of the U.S. Public Health Service since 1973, with headquarters in Atlanta; it was established in 1946 as the Communicable Disease Center. for confirmatory identification and molecular subtyping, but the initial clinical specimens were not sent along with the isolates. With few exceptions, clinical specimens available for analysis from these two patients and from other patients with inhalational anthrax were collected after initiation of antimicrobial therapy, resulting in few culture-positive results. For 3 of the 11 inhalational patients, laboratory confirmation was based on two of three available supportive tests, including PCR targeting two plasmid and one chromosomal target (14), immunohistochemistry or a reactive anti-protective antigen titer titer /ti·ter/ (ti´ter) the quantity of a substance required to react with or to correspond to a given amount of another substance. (immunoglobulin G immunoglobulin G n. Abbr. IgG The most abundant class of antibodies found in blood serum and lymph and active against bacteria, fungi, viruses, and foreign particles. Immunoglobulin G antibodies trigger action of the complement system. ELISA ELISA (e-li´sah) Enzyme-Linked Immuno-Sorbent Assay; any enzyme immunoassay using an enzyme-labeled immunoreactant and an immunosorbent. ELISA n. ) (12,20). Laboratory confirmation for the two cutaneous cases with skin biopsies Skin Biopsy Definition A skin biopsy is a procedure in which a small piece of living skin is removed from the body for examination, usually under a microscope, to establish a precise diagnosis. analyzed in this study was indeed achieved by these supportive laboratory tests: one case was confirmed by immunohistochemistry and a reactive anti-protective antigen title (IgG ELISA IgG ELISA, n.pr a diagnostic test for identifying reactive substances that provoke delayed hypersensitivity of the immune system. A solid-phase immunoassay that uses enzymes to test for IgG subclass reactions. ). For the other case all three supportive laboratory tests were positive. Previously, strains having <3% difference between their 16S rRNA genes were considered the same species (21). However, differences between 16S rRNA genes for some Bacillus species, such as B. anthracis, B. cereus, and B. thuringiensis, are <1% (1). Such small differences (e.g., one base between sequences or partial matches at a single nucleotide position in the 16S rRNA gene) have not been used for species differentiation. Our study clearly demonstrates that such small differences might be important for species identification. DNA-DNA hybridization DNA-DNA hybridization generally refers to a molecular biology technique that measures the degree of genetic similarity between pools of DNA sequences. It is usually used to determine the genetic distance between two species. and 16S rRNA sequencing studies have shown that these three Bacillus species are closely related and probably represent a single species (3,6,7). If the three were classified as a single species, 16S rRNA sequencing appears to have the potential to differentiate strains at the subspecies subspecies, also called race, a genetically distinct geographical subunit of a species. See also classification. level. Although pXO1 and pXO2 plasmids must be detected to confirm the virulence Virulence The ability of a microorganism to cause disease. Virulence and pathogenicity are often used interchangeably, but virulence may also be used to indicate the degree of pathogenicity. of B. anthracis, 16S rRNA sequencing has a powerful capacity to rapidly identify B. anthracis and other species. Although further studies are needed to fully evaluate 16S sequencing as a diagnostic assay Noun 1. diagnostic assay - an assay conducted for diagnostic purposes diagnostic test assay - a quantitative or qualitative test of a substance (especially an ore or a drug) to determine its components; frequently used to test for the presence or , its value as a tool for rapid initial screening in outbreak investigations has been demonstrated.
Table 1. Descriptions and GenBank accession numbers of 107 Bacillus
species strains analyzed in this study
GenBank
16S rRna
gene
accession
Species No. Identification number
B. anthracis Diversity
collection 18 2000031650 AY138379
2000031651 AY138372
2000031652 AY138374
2000031653 AY138373
2000031655 AY138376
2000031656 AY138375
2000031657 AY138382
2000031659 AY138377
2000031660 AY138378
2000031661 AY138369
2000031662 AY139368
2000031663 AY138381
2000031664 AY138383
2000031665 AY138366
2000031666 AY138371
2000031667 AY138380
2000031670 AY138367
2000031671 AY138370
Standard and
reference
strains 14 Ames AY138358
2002007651 AY138355
2002007650 AY138356
2002007649 AY138357
2000031887B AY138347
2000031666 AY138352
2000031368 AY138350
2000031244 AY138354
2000031078 AY138351
2000031076 AY138353
2000031259 AY138346
2000031075 AY138345
2000031887 AY138348
2000031136 AY 138349
Outbreak
strains 54 AY138291 to
AY138344
B. cereus 10 2000031486 AY138272
2000031491 AY138276
2000031498 AY138274
2000031503 AY138277
2000031513 AY138279
G3317 AY138278
G8639 AY138271
G9667 AY138273
H1439 AY138270
ATCC 14579 AY138275
B. thurin-
giensis 11 2000031482 AY138290
2000031485 AY138289
2000031494 AY138288
2000031496 AY138287
2000031508 AY138286
2000031509 AY138285
2002007400 AY138283
2002017401 AY138284
2000032755 AY138282
2000032757 AY138280
2000032756 AY138281
Geographic
or and
temporal
Species No. Identification origin (a)
B. anthracis Diversity
collection 18 2000031650 Human,
Turkey, 1991
2000031651 Bovine,
France, 1997
2000031652 Human, US, 1952
2000031653 Wool,
Pakistan, 1976
2000031655 Cow, China
2000031656 Ames
2000031657 Bovine
2000031659 Human,
Turkey, 1984
2000031660 Bovine,
US, 1937
2000031661 Human, South
Korea, 1994
2000031662 Zebra, Namibia
2000031663 Bovine, Poland
2000031664 Porcine,
German, 1971
2000031665 Bovine,
Argentina
2000031666 UK
2000031667 Sheep,
Italy, 1994
2000031670 Human,
Turkey, 1985
2000031671 Bovine, Zambia
Standard and
reference
strains 14 Ames Ames
2002007651 Sterne, Chile
2002007650 Sterne, Chile
2002007649 Pasteur, Chile
2000031887B Vaccine
2000031666 Vollum
2000031368 Vollum
2000031244 Vollum
2000031078 Vollum M36
2000031076 Vollum
2000031259 Pasteur
2000031075 Sterne
2000031887 V770-NP1-R
2000031136 New Hampshire
Outbreak
strains 54 US Oct/Dec 2001
B. cereus 10 2000031486 Human, US, 1994
2000031491 Human, US, 1997
2000031498 Human, US, 1979
2000031503 Human, US, 1999
2000031513 Human, US, 1986
G3317 Human,
Israel, 1989
G8639 Milk,
Bolivia, 1993
G9667 Human, US, 1995
H1439 Human, US, 2000
ATCC 14579 1887
B. thurin-
giensis 11 2000031482 Human, US, 1989
2000031485 Spray, US, 1993
2000031494 Human, US, 1985
2000031496 Human, US, 1981
2000031508 Human, US, 1985
2000031509 Human, US, 1985
2002007400 Powder,
US, 2001
2002017401 Powder,
US, 2001
2000032755 Environment,
US, 2000
2000032757 Environment,
US, 2000
2000032756 Human, US,
1981
16S rRNA
Species No. Identification type
B. anthracis Diversity
collection 18 2000031650 6
2000031651 6
2000031652 6
2000031653 6
2000031655 6
2000031656 6
2000031657 6
2000031659 6
2000031660 6
2000031661 6
2000031662 6
2000031663 6
2000031664 6
2000031665 6
2000031666 6
2000031667 6
2000031670 6
2000031671 6
Standard and
reference
strains 14 Ames 6
2002007651 6
2002007650 6
2002007649 6
2000031887B 6
2000031666 6
2000031368 6
2000031244 6
2000031078 6
2000031076 6
2000031259 6
2000031075 6
2000031887 6
2000031136 6
Outbreak
strains 54 6
B. cereus 10 2000031486 12
2000031491 7
2000031498 9
2000031503 7
2000031513 13
G3317 7
G8639 3
G9667 12
H1439 2
ATCC 14579 9
B. thurin-
giensis 11 2000031482 10
2000031485 10
2000031494 10
2000031496 10
2000031508 10
2000031509 10
2002007400 10
2002017401 10
2000032755 10
2000032757 10
2000032756 10
MLVA
Species No. Identification genotype (b)
B. anthracis Diversity
collection 18 2000031650 23
2000031651 80
2000031652 68
2000031653 69
2000031655 57
2000031656 62
2000031657 10
2000031659 28
2000031660 25
2000031661 34
2000031662 35
2000031663 15
2000031664 38
2000031665 45
2000031666 77
2000031667 20
2000031670 41
2000031671 30
Standard and
reference
strains 14 Ames 62
2002007651 ND
2002007650 ND
2002007649 ND
2000031887B ND
2000031666 77
2000031368 77
2000031244 *
2000031078 ND
2000031076 *
2000031259 **
2000031075 *
2000031887 *
2000031136 73
Outbreak
strains 54 62
B. cereus 10 2000031486 NA
2000031491 NA
2000031498 NA
2000031503 NA
2000031513 NA
G3317 NA
G8639 NA
G9667 NA
H1439 NA
ATCC 14579 NA
B. thurin-
giensis 11 2000031482 NA
2000031485 NA
2000031494 NA
2000031496 NA
2000031508 NA
2000031509 NA
2002007400 NA
2002017401 NA
2000032755 NA
2000032757 NA
2000032756 NA
(a) Date and source of isolation are provided when available; *,
lacking pXO2; **, lacking pXO1.
(b) ND, MLVA (8).
NA, not applicable.
Table 2. Primers used for amplification and sequencing of the 16S
rRNA gene of Bacillus anthracis, B. thuringiensis, and B. cereus (a)
Generic primers used for 16S rRNA amplification
8F 5'AGT TGA TCC TGG CTC AG 3'
1492R 5'ACC TTG TTA CGA CTT3'
Primers for amplification of the 16S rRNA gene
67F 5'TGA AAA CTG AAC GAA ACA AAC 3'
1671R 5'CTC TCA AAA CTG AAC AAA ACG AAA 3'
Inner primers used for nested PCR on clinical samples
23F 5'ACA AAC AAC GTG AAA CGT CAA 3'
136R 5'AAA CGA AAC ACG GAA ACT T 3'
Primers used for sequencing of the 16S rRNA gene
104F 5'GGA CGG GTG AGT AAC ACG TG 3'
104R 5'CAC GTG TTA CTC ACC CGT CC 3'
1230F 5'TAC ACA CGT GCT ACA ATG 3'
1390F 5'GGG CCT TGT ACA CAC CG 3'
1390R 5'CGG TGT GTA CAA GGC CC 3'
8F 5'AGT TGA TCC TGG CTC AG 3'
357F 5'TAC GGG AGG CAG CAG 3'
357R 5'CTG CTG CCT CCC GTA 3'
530F 5'CAG CAG CCG CGG TAA TAC 3'
530R 5'GTA TTA CCG CGG CTG CTG 3'
790F 5'ATT AGA TAC CCT GGT AG 3'
790R 5'CTA CCA GGG TAT CTA AT 3'
981F 5'CCC GCA ACG AGC GCA ACC C 3'
981R 5'GGG TTG CGC TCG TTG CGG G 3'
(a) Primers 67F and 1671R or primers 23F and 136R were also used for
16S sequence on isolates or clinical samples, respectively.
Table 3. 16S rRNA gene types identified among 125 Bacillus spp.
strains analyzed in this study (n=107) and available at GenBank (n=18)
Positions (a)
1 2 3
Bacillus No. of
16S type species strains (77) (90) (92)
16S type identified in 107 strains in this study
2 cereus 1 R (b) Y W
3 cereus 1 G C A
6 anthracis 86 A T T
7 cereus 3 A T T
9 cereus 2 A T T
10 thuringiensis 11 A T T
12 cereus 2 A T T
13 cereus 1 A T T
16S type identified in strains available at GenBank (d)
1 mycoides 2 A T T
4 thuringiensis 3 G C A
5 cereus 8 G C A
7 anthracis 3 A T T
Positions (a)
4 5 6 7 8 9
16S type (182) (189) (192) (200) (208) (1,015)
16S type identified in 107 strains in this study
2 C (c) A T T G A
3 Y A T T G A
6 C A C T G C
7 C A C T G C
9 C A C T G A
10 C A Y T G A
12 Y A T T G A
13 C A C T G C
16S type identified in strains available at GenBank (d)
1 C C C G C C
4 C A T T G A
5 C A C T G A
7 C A C T G C
Positions (a)
10 11 12 13
16S type (1,036) (1,045) (1,146) (1,462)
16S type identified in 107 strains in this study
2 T A A A
3 T A A A
6 T A W T
7 T A A T
9 T A A T
10 T A A T
12 T A A T
13 T A T T
16S type identified in strains available at GenBank (d)
1 C G A -- (e)
4 T A A --
5 T A A --
7 T A A T
(a) Numbers refer to the number of positions where mismatches are
found. Numbers in parentheses refer to positions in the 16S rRNA gene.
(b) R refers to a purine (A or G) at that position; Y refers to a
pyrimidine (C or T) at that position; and W refers to an A or T at that
position.
(c) A, C, G, and T refer to the four deoxynucleotides that DNA
comprises.
(d) Five additional positions of differences (positions 5, 7, 8, 10,
and 11) were found when GenBank sequences were used.
(e) The last position (position 13) on 16S types 1, 4, and 5 is missing
because those GenBank sequences are shorter.
Table 4. Results of laboratory testing on seven clinical samples in
which 16S type 6 was identified
Patient
ID (a) Diagnosis Laboratory confirmation (b)
2i Inhalational anthrax IHC + PCR of pleural fluid; serology
10i Inhalational anthrax B. anthracis isolated from blood and
pleural fluid
11i Inhalational anthrax B. anthracis isolated from blood
7c Cutaneous anthrax IHC + PCR on skin biopsy
Clinical specimens
ID (a) Type Culture Bacillus anthracis-
specific PCR (b)
2i Tissue Neg Neg
10i Pleural fluid Neg Pos
Pleural fluid Neg Pos
Blood Neg ND
Lymph node Neg Pos
11i Lymph node Neg Pos
7c Skin from forehead Neg Pos
(a) Patient identification numbers are described in references 12 and
13; I, inhalational case; C, cutaneous case; PCR, polymerase chain
reaction.
(b) The immunohistochemical (IHC), serologic, and PCR results are
described in reference 14.
Acknowledgement Arijana Boras Bo·rås A city of southwest Sweden east of Göteborg. It was founded in 1632. Population: 60,900. was a Fellow of the International Emerging Infectious Diseases An emerging infectious disease (EID) is an infectious disease whose incidence has increased in the past 20 years and threatens to increase in the near future. EIDs include diseases caused by a newly identified microorganism or newly identified strain of a known microorganism (e.g. Fellowship Program administered by the Association of Public Health Laboratories The Association of Public Health Laboratories (APHL) works to safeguard the public's health by strengthening government laboratories with a public health mandate in the United States and across the world. (APHL APHL Association of Public Health Laboratories ) and the CDC See Control Data, century date change and Back Orifice. CDC - Control Data Corporation Foundation and funded by an Educational Grant from Eli Lilly and Company Eli Lilly and Company (NYSE: LLY) is a global pharmaceutical company and one of the world's largest corporations. Eli Lilly's global headquarters is located in Indianapolis, Indiana, in the United States. . Dr. Sacchi is a research microbiologist in the Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic mycotic /my·cot·ic/ (mi-kot´ik) 1. pertaining to mycosis. 2. caused by a fungus. my·cot·ic adj. 1. Relating to mycosis. 2. Diseases, National Center for Infectious Diseases infectious diseases: see communicable diseases. , CDC, and the Adolfo Lutz Adolfo Lutz was a Brazilian physician, 1855-1940, father of tropical medicine and medical zoology in Brazil, and a pioneer epidemiologist and researcher in infectious diseases. Lutz was born in Rio de Janeiro, on December 18, 1855, to a family of Swiss origins. Institute, Sao Paulo, Brazil. His research interests include Neisseria meningitidis Neisseria men·in·git·i·dis n. The bacteria that is the causative agent of cerebrospinal meningitis; meningococcus. Neisseria meningitidis disease and the molecular biology molecular biology, scientific study of the molecular basis of life processes, including cellular respiration, excretion, and reproduction. The term molecular biology was coined in 1938 by Warren Weaver, then director of the natural sciences program at the Rockefeller of pathogenic bacteria Pathogenic bacteria Bacteria that produce illness. Mentioned in: Gastroenteritis . References (1.) Thorne CB. In: Sonenshein AL, Hock hock: see wine. JA, Losick R. Bacillus subtilis Noun 1. Bacillus subtilis - a species of bacillus found in soil and decomposing organic matter; some strains produce antibiotics Bacillus globigii, grass bacillus, hay bacillus and other gram-positive bacteria. Washington: American Society for Microbiology The American Society for Microbiology (ASM) is a scientific organization, based in the United States although with over 43,000 members throughout the world. It is the largest single life science professional organization and its members include those whose interests encompass basic ; 1993. p. 113-24. (2.) Ash C, Farrow farrow see farrowing. JAE JAE Journal of Applied Econometrics JAE Journal of Architectural Education JAE Journal of Aesthetic Education JAE Jump If Above or Equal JAE Journal of Architectural Engineering JAE Java Application Environment JAE Junta Autónoma de Estradas , Wallbanks S, Collins MD. Phylogenetic phy·lo·ge·net·ic adj. 1. Of or relating to phylogeny or phylogenetics. 2. Relating to or based on evolutionary development or history. heterogeneity of the Bacillus revealed by comparative analysis of small subunit sub·u·nit n. A subdivision of a larger unit. Noun 1. subunit - a monetary unit that is valued at a fraction (usually one hundredth) of the basic monetary unit fractional monetary unit ribosomal RNA ribosomal RNA n. See rRNA. ribosomal RNA (rī´bōsō´m sequences. Lett Appl Microbiol 1991; 13:202-6. (3.) Ash C, Farrow JA, Dorsch M, Stackebrandt E, Collins MD. Comparative analysis of Bacillus anthracis, Bacillus cereus Bacillus ce·re·us n. A species of Bacillus that causes an emetic type and a diarrheal type of food poisoning in humans. , and related species on the basis of reverse transcriptase Reverse transcriptase Any of the deoxyribonucleic acid (DNA) polymerases present in particles of retroviruses which are able to carry out DNA synthesis using an RNA template. sequencing of 16S rRNA. Int J Syst Bacteriol 1991;41:343-6. (4.) Helgason E, Okstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, et al. Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence. Appl Environ Microbiol 2000;66:2627-30. 5. Kaneko T, Nozaki R, Aizawa K. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol Immunol 1978;22:639-41. (6.) Seki T, Chung C, Mikami H, Oshima Y. Deoxyribonucleic acid homology homology (hōmŏl`əjē), in biology, the correspondence between structures of different species that is attributable to their evolutionary descent from a common ancestor. and taxonomy taxonomy: see classification. taxonomy In biology, the classification of organisms into a hierarchy of groupings, from the general to the particular, that reflect evolutionary and usually morphological relationships: kingdom, phylum, class, order, of the genus Bacillus Noun 1. genus Bacillus - type genus of the Bacillaceae; includes many saprophytes important in decay of organic matter and a number of parasites B, bacillus - aerobic rod-shaped spore-producing bacterium; often occurring in chainlike formations; found primarily in . Int J Syst Bacteriol 1978;28:182-9. (7.) Somerville HJ, Jones ML. DNA competition studies within the Bacillus cereus group of bacilli bacilli /ba·cil·li/ (bah-sil´i) plural of bacillus. bacilli see bacillus. . J Gen Microbiol 1972;73:257-65. (8.) Keim P, Price LB, Klevytska AM, Smith KL, Schupp JM, Okinaka R, et al. Multiple-locus variable-number tandem repeat This is a term from genetics, which describes a pattern that helps determine an individual's inherited traits. Tandem repeats and variable number tandem repeats in DNA occur when a pattern of two or more nucleotides is repeated and the repetitions are directly adjacent to analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol 2000;182:2928-36. (9.) Khan AS, Morse S, Lillibridge S. Public-health preparedness for biological terrorism Noun 1. biological terrorism - terrorism using the weapons of biological warfare bioterrorism act of terrorism, terrorism, terrorist act - the calculated use of violence (or the threat of violence) against civilians in order to attain goals that are in the USA. Lancet 2000;356:1179-82. (10.) Logan NA, Turnbull PCB PCB: see polychlorinated biphenyl. PCB in full polychlorinated biphenyl Any of a class of highly stable organic compounds prepared by the reaction of chlorine with biphenyl, a two-ring compound. . In: Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH, editors. Manual of clinical microbiology Clinical microbiology The adaptation of microbiological techniques to the study of the etiological agents of infectious disease. Clinical microbiologists determine the nature of infectious disease and test the ability of various antibiotics to inhibit or kill . 7th ed. Washington: American Society for Microbiology; 1999. p. 357-69. (11.) Eden PA, Schmidt TM, Akemore RE Pace NR. Phylogenetic analysis of Aquaspirillum magnetotacticum using polymerase chain reaction-amplified 16S rRNA-specific DNA. Int J Syst Bacteriol 1991;41:324-5. (12.) Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, et al. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 2001;7:933-44. (13.) Barakat LA, Quentzel HL, Jernigan JA, Kirschke DL, Griffith K, Spear SM, et al. Fatal inhalational anthrax in a 94-year-old Connecticut woman. JAMA JAMA abbr. Journal of the American Medical Association 2002;287:863-8. (14.) Hoffmaster AR, Meyer RF, Bowen M, Marston CK, Weyant RS, Barnett GA, et al. Evaluation and validation of a real-time PCR assay for rapid identification of Bacillus anthracis. Emerg Infect Dis 2002;8:1111-6. (15.) Ticknor LO, Kolsto AB, Hill KK, Keim P, Laker lak·er n. 1. A fish, such as the lake trout, that lives in a lake. 2. A ship used on lakes. MT, Tonks Tonks may refer to:
Amplified Fragment Length Polymorphism (AFLP analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol 2001;67:4863-73. (16.) Nubel U, Engelen B, Felske A, Snaidr J, Wieshuber A, Amann RI, et al. Sequence heterogeneities of genes encoding See encode. 16S rRNAs in Paenibacillus polymyxa Paenibacillus polymyxa is a Gram-positive bacterium used as a soil inoculant in agriculture and horticulture. It is capable of fixing nitrogen. detected by temperature gradient gel electrophoresis Temperature gradient gel electrophoresis (TGGE) is a form of electrophoresis where there is a temperature gradient across the gel. TGGE is useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins. . J Bacteriol 1996;178:5636-43. (17.) Rainey FA, Ward-Rainey NL, Janssen PH, Hippe H, Stackebrandt E. Clostridium clostridium Any of the rod-shaped, usually gram-positive bacteria (see gram stain) that make up the genus Clostridium. They are found in soil, water, and the intestinal tracts of humans and other animals. Some species grow only in the complete absence of oxygen. paradoxum DSM 1. DSM - Data Structure Manager. An object-oriented language by J.E. Rumbaugh and M.E. Loomis of GE, similar to C++. It is used in implementation of CAD/CAE software. DSM is written in DSM and C and produces C as output. 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences intervening sequence see intron. . Microbiology 1996;142:2087-95. (18.) Liefting LW, Andersen MT, Beever RE, Gardner RC, Forster RL. Sequence heterogeneity in the two 16S rRNA genes of Phormium Phor´mi`um n. 1. (Bot.) A genus of liliaceous plants, consisting of one species (Phormium tenax). See Flax-plant. yellow leaf phytoplasma. Appl Environ Microbiol 1996;62:3133-9. (19.) Mylvaganam S, Dennis PP. Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic halophilic pertaining to or characterized by an affinity for salt; requiring a high concentration of salt for optimal growth. archaebacterium ar·chae·bac·te·ri·um n. pl. ar·chae·bac·te·ri·a An archaeon. [archae(o)- + bacterium. Haloarcula marismortui. Genetics 1992; 130:399-410. (20.) Centers for Disease Control and Prevention. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR MMWR Morbidity & Mortality Weekly Report Epidemiology A news bulletin published by the CDC, which provides epidemiologic data–eg, statistics on the incidence of AIDS, rabies, rubella, STDs and other communicable diseases, causes of mortality–eg, Morb Mortal Wkly Rep 2001;50:889-93. (21.) Stackebrandt E, Goebel BM. Taxonomic tax·o·nom·ic also tax·o·nom·i·cal adj. Of or relating to taxonomy: a taxonomic designation. tax note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species in bacteriology bacteriology Study of bacteria. Modern understanding of bacterial forms dates from Ferdinand Cohn's classifications. Other researchers, such as Louis Pasteur, established the connection between bacteria and fermentation and disease. . Int J Syst Bacteriol 1994;44:846-9. Address for correspondence: Claudio T. Sacchi, Meningitis and Special Pathogens Branch, Centers for Disease Control and Prevention, 1600 Clifton Rd., N.E., Mailstop D11, Atlanta, GA 30333, USA; fax: 404-639-4421; e-mail: cls9@cdc.gov Claudio T. Sacchi, * ([dagger]) Anne M. Whitney, * Leonard W. Mayer, * Roger Mercy, * Arnold Steigerwalt, * Arijana Boras, * ([double dagger double dagger n. A reference mark (  ) used in printing and writing. Also called diesis.Noun 1. ]) Robin S. Weyant, * and Tanja Popovic * * Centers for Disease Control and Prevention, Atlanta, Georgia, USA; ([dagger]) Adolfo Lutz Institute, Sao Paulo, Brazil; and ([double dagger]) University Hospital for Infectious Diseases, Zagreb, Croatia |
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