Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates

BACKGROUND: Bacillus anthracis and Bacillus cereus can usually be distinguished by standard microbiological methods (e.g., motility, hemolysis, penicillin susceptibility and susceptibility to gamma phage) and PCR. However, we have identified 23 Bacillus spp. isolates that gave discrepant results when assayed by standard microbiological methods and PCR. We used multiple-locus variable-number tandem repeat analysis (MLVA), multiple-locus sequence typing (MLST), and phenotypic analysis to characterize these isolates, determine if they cluster phylogenetically and establish whether standard microbiological identification or PCR were associated with false positive/negative results. RESULTS: Six isolates were LRN real-time PCR-positive but resistant to gamma phage; MLVA data supported the identification of these isolates as gamma phage-resistant B. anthracis. Seventeen isolates were LRN real-time PCR-negative but susceptible to gamma phage lysis; these isolates appear to be a group of unusual gamma phage-susceptible B. cereus isolates that are closely related to each other and to B. anthracis. All six B. anthracis MLVA chromosomal loci were amplified from one unusual gamma phage-susceptible, motile, B. cereus isolate (although the amplicons were atypical sizes), and when analyzed phylogenetically, clustered with B. anthracis by MLST. CONCLUSION: MLVA and MLST aided in the identification of these isolates when standard microbiological methods and PCR could not definitely identify or rule out B. anthracis. This study emphasized the need to perform multiple tests when attempting to identify B. anthracis since relying on a single assay remains problematic due to the diverse nature of bacteria

Molecular Subtyping of Bacillus anthracis and the 2001 Bioterrorism-Associated Anthrax Outbreak, United States

Molecular subtyping of Bacillus anthracis played an important role in differentiating and identifying anthrax strains during the 2001 bioterrorism-associated outbreak. Because B. anthracis has a low level of genetic variability, only a few subtyping methods, with varying reliability, exist. We initially used multiple-locus variable-number tandem repeat analysis (MLVA) to subtype 135 B. anthracis isolates associated with the outbreak. All isolates were determined to be of genotype 62, the same as the Ames strain used in laboratories. We sequenced the protective antigen gene (pagA) from 42 representative outbreak isolates and determined they all had a pagA sequence indistinguishable from the Ames strain (PA genotype I). MLVA and pagA sequencing were also used on DNA from clinical specimens, making subtyping B. anthracis possible without an isolate. Use of high-resolution molecular subtyping determined that all outbreak isolates were indistinguishable by the methods used and probably originated from a single source. In addition, subtyping rapidly identified laboratory contaminants and non-outbreak–related isolates

Molecular Characterization of Corynebacterium diphtheriae isolates, Russia, 1957–1987

In the 1990s, the Newly Independent and Baltic States of the former Soviet Union experienced the largest diphtheria outbreak since the 1960s; it was caused by Corynebacterium diphtheriae strains of a unique clonal group. To address its origin, we studied 47 clinical isolates from Russia and demonstrated that this clonal group was an integral part of the endemic reservoir that existed in Russia at least 5 years before the epidemic began

Earthworms (Annelida: Oligochaeta, Lumbricidae) of the Kopaonik National Park (Serbia)

This paper presents the first list of earthworm fauna of the Kopaonik National Park, a total of 16 taxa, belonging to eight genera of the family Lumbricidae. The genera with the largest number of the registered taxa are Dendrobaena and Lumbricus, while the genera Aporrectodea, Bimastos, Eisenia, Eiseniella, Octodrilus and Octolasion are represented by one species each. Also, the review of zoogeographical types and ecological categories of registered species are presented. Half of the species are peregrine, while the rest are autochthonous. Regarding ecological categories, anecic (2) and endogeic (4) species are more sensitive to high mountain environmental conditions than epigeic (12) species, which are more adapted to such conditions. Overall, our results highlight how little was previously known about the earthworm fauna in this area and emphasize the need for further collecting to better understand the hidden earthworm diversity in Kopaonik NP.Publishe

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

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 generom 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 seqs fuences 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

Bioterrorism-Related Anthrax: International Response by the Centers for Disease Control and Prevention

After reports of the intentional release of Bacillus anthracis in the United States, epidemiologists, laboratorians, and clinicians around the world were called upon to respond to widespread political and public concerns. To respond to inquiries from other countries regarding anthrax and bioterrorism, the Centers for Disease Control and Prevention established an international team in its Emergency Operations Center. From October 12, 2001, to January 2, 2002, this team received 130 requests from 70 countries and 2 territories. Requests originated from ministries of health, international organizations, and physicians and included subjects ranging from laboratory procedures and clinical evaluations to assessments of environmental and occupational health risks. The information and technical support provided by the international team helped allay fears, prevent unnecessary antibiotic treatment, and enhance laboratory-based surveillance for bioterrorism events worldwide

Leptospirosis in “Eco-Challenge” Athletes, Malaysian Borneo, 2000

Adventure travel is becoming more popular, increasing the likelihood of contact with unusual pathogens. We investigated an outbreak of leptospirosis in “Eco-Challenge” multisport race athletes to determine illness etiology and implement public health measures. Of 304 athletes, we contacted 189 (62%) from the United States and 26 other countries. Eighty (42%) athletes met our case definition. Twenty-nine (36%) case-patients were hospitalized; none died. Logistic regression showed swimming in the Segama River (relative risk [RR]=2.0; 95% confidence interval [CI]=1.3 to 3.1) to be an independent risk factor. Twenty-six (68%) of 38 case-patients tested positive for leptospiral antibodies. Taking doxycycline before or during the race was protective (RR=0.4, 95% CI=0.2 to 1.2) for the 20 athletes who reported using it. Increased adventure travel may lead to more frequent exposure to leptospires, and preexposure chemoprophylaxis for leptospirosis (200 mg oral doxycycline/week) may decrease illness risk. Efforts are needed to inform adventure travel participants of unique infections such as leptospirosis

Bacillus anthracis Aerosolization Associated with a Contaminated Mail Sorting Machine

On October 12, 2001, two envelopes containing Bacillus anthracis spores passed through a sorting machine in a postal facility in Washington, D.C. When anthrax infection was identified in postal workers 9 days later, the facility was closed. To determine if exposure to airborne B. anthracis spores continued to occur, we performed air sampling around the contaminated sorter. One CFU of B. anthracis was isolated from 990 L of air sampled before the machine was activated. Six CFUs were isolated during machine activation and processing of clean dummy mail. These data indicate that an employee working near this machine might inhale approximately 30 B. anthracis-containing particles during an 8-h work shift. What risk this may have represented to postal workers is not known, but the risk is approximately 20-fold less than estimates of sub-5 micron B. anthracis-containing particles routinely inhaled by asymptomatic, unvaccinated workers in a goat-hair mill

Two-Component Direct Fluorescent-Antibody Assay for Rapid Identification of Bacillus anthracis

A two-component direct fluorescent-antibody (DFA) assay, using fluorescein-labeled monoclonal antibodies specific to the Bacillus anthracis cell wall (CW-DFA) and capsule (CAP-DFA) antigens, was evaluated and validated for rapid identification of B. anthracis. We analyzed 230 B. anthracis isolates; 228 and 229 were positive by CW-DFA and CAP-DFA assays, respectively. We also tested 56 non–B. anthracis strains; 10 B. cereus and 2 B. thuringiensis were positive by the CW-DFA assay, and 1 B. megaterium strain was positive by CAP-DFA. Analysis of the combined DFA results identified 227 of 230 B. anthracis isolates; all 56 strains of the other Bacillus spp. were negative. Both DFA assays tested positive on 14 of 26 clinical specimens from the 2001 anthrax outbreak investigation. The two-component DFA assay is a sensitive, specific, and rapid confirmatory test for B. anthracis in cultures and may be useful directly on clinical specimens

Surface Sampling Methods for Bacillus anthracis Spore Contamination

During an investigation conducted December 17–20, 2001, we collected environmental samples from a U.S. postal facility in Washington, D.C., known to be extensively contaminated with Bacillus anthracis spores. Because methods for collecting and analyzing B. anthracis spores have not yet been validated, our objective was to compare the relative effectiveness of sampling methods used for collecting spores from contaminated surfaces. Comparison of wipe, wet and dry swab, and HEPA vacuum sock samples on nonporous surfaces indicated good agreement between results with HEPA vacuum and wipe samples. However, results from HEPA vacuum sock and wipe samples agreed poorly with the swab samples. Dry swabs failed to detect spores >75% of the time they were detected by wipe and HEPA vacuum samples. Wipe samples collected after HEPA vacuum samples and HEPA vacuum samples after wipe samples indicated that neither method completely removed spores from the sampled surfaces