Bacillus anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

Abstract

In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.Long considered a biowarfare agent, Bacillus anthracis was used in 2001 in an act of bioterrorism. After bacterial endospores were placed into envelopes and delivered by the United States postal system to unsuspecting victims, 22 people were infected of whom 11 developed inhalational anthrax and 5 died (14, 15). As a result, first responders delivered hundreds of thousands of environmental specimens to laboratories across the nation that are part of the National Laboratory Response Network (LRN), a coordinated system of sentinel, reference, and national laboratories established by the Centers for Disease Control and Prevention (CDC) (6, 7). Of the three Florida Department of Health (FDOH) laboratories with biosafety level 3 facilities designated to receive these specimens, the FDOH Tampa Laboratory received and analyzed 1,046 environmental samples from first responders across west-central Florida over the last 3 months of 2001. Among these specimens, 19 loose powders or swab samples of powders that were brought in by local law enforcement officers and considered plausible threats initially tested positive for potentially carrying a B. anthracis isolate.Because of the initial positive results, the 19 powders and swabs were cultured and all isolates were tested further using the LRN tests, consisting of nonspecific phenotypic physical/ biochemical tests and specific molecular methods for B. anthracis such as gamma phage susceptibility testing, cell wall and capsule detection by direct fluorescence antibody tests (DFA), and amplification of targeted DNA sequences by PCR (31). After more tests...

Detection of Coxiella burnetii in Complex Matrices by Using Multiplex Quantitative PCR during a Major Q Fever Outbreak in The Netherlands

“…The amplification of multiple signature sequences per organism also will reduce false-positive results in complex samples. False positives can be an issue if detection relies on single targets due to the presence of homologous sequences in related organisms or unknown sources when analyzing environmental samples (19,20).…”

A Bacillus anthracis -Based In Vitro System Supports Replication of Plasmid pXO2 as Well as Rolling-Circle-Replicating Plasmids

“…The pAM␤1 family of replicons requires transcription into the ori from their rep gene for replication to occur (9). Furthermore, rep gene transcription is regulated by CT-driven attenuation (7,31). We performed Northern blot analysis to identify the repS transcript and/or its attenuated products.…”

Development of Three Real-Time PCR assays to Detect Bacillus anthracis and Assessment of Diagnostic Utility

“…pXO1 and pXO2 plasmids and/or elements have been identified within the genomes of other members of the genus Bacillus [10]. In silico (BLAST) analysis of the pXO1 and pXO2 PCR amplicon sequences indicated that if tested against the B. cereus G9241 strain [8] then a pXO1 positive would result, as would a pXO2 PCR if tested against the B. cereus (var.)…”

“…• mL -1 (4 cases) and 10 1 cfu • mL -1 (2 cases). Bloods were taken at 12 hour intervals after inoculation and in some cases the onset of bacteraemia was not immediately associated with observable disease symptoms.…”

“…However, these plasmids (or elements originating from these plasmids) have been found in other Bacillus spp. [8][9][10] so the provision of a chromosomal assay, specific to B. anthracis, can help distinguish classical B. anthracis strains from other pXO1 and pXO2 harbouring species. In this paper we describe the development of three real-time PCRs to detect different genetic regions of B. anthracis (pXO1, pXO2 and chromosomal targets) and assess the diagnostic utility of two of these PCRs from samples obtained from three B. anthracis murine infection models.…”