The clinical laboratory diagnosis of cutaneous anthrax is generally established by conventional microbiological methods, such as culture and directly straining smears of clinical specimens. However, these methods rely on recovery of viable Bacillus anthracis cells from swabs of cutaneous lesions and often yield negative results. This study developed a rapid protocol for detection of B. anthracis on clinical swabs. Three types of swabs, flocked-nylon, rayon, and polyester, were evaluated by 3 extraction methods, the swab extraction tube system (SETS), sonication, and vortex. Swabs were spiked with virulent B. anthracis cells, and the methods were compared for their efficiency over time by culture and real-time PCR. Viability testing indicated that the SETS yielded greater recovery of B. anthracis from 1-day-old swabs; however, reduced viability was consistent for the 3 extraction methods after 7 days and nonviability was consistent by 28 days. Real-time PCR analysis showed that the PCR amplification was not impacted by time for any swab extraction method and that the SETS method provided the lowest limit of detection. When evaluated using lesion swabs from cutaneous anthrax outbreaks, the SETS yielded culture-negative, PCR-positive results. This study demonstrated that swab extraction methods differ in their efficiency of recovery of viable B. anthracis cells. Furthermore, the results indicated that culture is not reliable for isolation of B. anthracis from swabs at ≥7 days. Thus, we recommend the use of the SETS method with subsequent testing by culture and real-time PCR for diagnosis of cutaneous anthrax from clinical swabs of cutaneous lesions.
Secondary cell wall polysaccharides (SCWPs) are important structural components of the Bacillus cell wall and contribute to the array of antigens presented by these organisms in both spore and vegetative forms. We previously found that antisera raised to Bacillus anthracis spore preparations cross-reacted with SCWPs isolated from several strains of pathogenic B. cereus, but did not react with other phylogenetically related but nonpathogenic Bacilli, suggesting that the SCWP from B. anthracis and pathogenic B. cereus strains share specific structural features. In this study, SCWPs from three strains of B. cereus causing severe or fatal pneumonia (G9241, 03BB87 and 03BB102) were isolated and subjected to structural analysis and their structures were compared to SCWPs from B. anthracis. Complete structural analysis was performed for the B. cereus G9241 SCWP using NMR spectroscopy, mass spectrometry and derivatization methods. The analyses show that SCWPs from B. cereus G9241 has a glycosyl backbone identical to that of B. anthracis SCWP, consisting of multiple trisaccharide repeats of: →6)-α-d-GlcpNAc-(1 → 4)-β-d-ManpNAc-(1 → 4)-β-d-GlcpNAc-(1→. Both the B. anthracis and pathogenic B. cereus SCWPs are highly substituted at all GlcNAc residues with α- and β-Gal residues, however, only the SCWPs from B. cereus G9241 and 03BB87 carry an additional α-Gal substitution at O-3 of ManNAc residues, a feature lacking in the B. anthracis SCWPs. Both the B. anthracis and B. cereus SCWPs are pyruvylated, with an approximate molecular mass of ≈12,000 Da. The implications of these findings regarding pathogenicity and cell wall structure are discussed.
Bacillus anthracis; Bacillus cereus; cell wall; polysaccharide; structure
To determine potential links between the clinical isolate to animal products and their geographic origin, we genotyped (MLVA-8, MVLA-15, and canSNP analysis) 80 environmental and 12 clinical isolates and 2 clinical specimens from five cases of anthrax (California in 1976 [n = 1], New York in 2006 [n = 1], Connecticut in 2007 [n = 2], and New Hampshire in 2009[n = 1]) resulting from recreational handling of animal products. For the California case, four clinical isolates were identified as MLVA-8 genotype (GT) 76 and in the canSNP A.Br.Vollum lineage, which is consistent with the Pakistani origin of the yarn. Twenty eight of the California isolates were in the A.Br.Vollum canSNP lineage and one isolate was in the A.Br. 003/004 canSNP sub-group. All 52 isolates and both clinical specimens related to the New York and Connecticut cases were MLVA-8 GT 1. The animal products associated with the NY and CT cases were believed to originate from West Africa, but no isolates from this region are available to be genotyped for comparison. All isolates associated with the New Hampshire case were identical and had a new genotype (GT 149). Isolates from the NY, CT and NH cases diverge from the established canSNP phylogeny near the base of the A.Br.011/009. This report illustrates the power of the current genotyping methods and the dramatically different epidemiological conditions that can lead to infections (i.e., contamination by a single genotype versus widespread contamination of numerous genotypes). These cases illustrate the need to acquire and genotype global isolates so that accurate assignments can be made about isolate origins.
Cutaneous anthrax outbreaks occurred in Bangladesh from August to October 2009. As part of the epidemiological response and to confirm anthrax diagnoses, serum samples were collected from suspected case patients with observed cutaneous lesions. Anthrax lethal factor (LF), anti-protective antigen (anti-PA) immunoglobulin G (IgG), and anthrax lethal toxin neutralization activity (TNA) levels were determined in acute and convalescent serum of 26 case patients with suspected cutaneous anthrax from the first and largest of these outbreaks. LF (0.005–1.264 ng/mL) was detected in acute serum from 18 of 26 individuals. Anti-PA IgG and TNA were detected in sera from the same 18 individuals and ranged from 10.0 to 679.5 μg/mL and 27 to 593 units, respectively. Seroconversion to serum anti-PA and TNA was found only in case patients with measurable toxemia. This is the first report of quantitative analysis of serum LF in cutaneous anthrax and the first to associate acute stage toxemia with subsequent antitoxin antibody responses.
Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic γ-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean ± standard error [SE] between animals) were low at 24 h postchallenge (0.03 ± 1.82 ng/ml), increased at 48 h to 39.53 ± 0.12 ng/ml (phase 1), declined at 72 h to 13.31 ± 0.24 ng/ml (phase 2), and increased at 96 h (82.78 ± 2.01 ng/ml) and 120 h (185.12 ± 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 ± 2 ng/ml), declined at 72 h (14 ± 0.2 ng/ml), and then increased at 96 h (3,401 ± 8 ng/ml) and 120 h (6,004 ± 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% ± 0.13%) to 48 h (75.6% ± 0.08%) and declined at 72 h (62.4% ± 0.05%). The 72-h declines may establish a “go/no go” turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.
Bacillus cereus is most commonly associated with foodborne illness (diarrheal and emetic) but is also an opportunistic pathogen that can cause severe and fatal infections. Several multilocus sequence typing (MLST) schemes have recently been developed to genotype B. cereus and analysis has suggested a clonal or weakly clonal population structure for B. cereus and its close relatives B. anthracis and B. thuringiensis. In this study we used MLST to determine if B. cereus isolates associated with illnesses of varying severity (e.g., severe, systemic vs. gastrointestinal (GI) illness) were clonal or formed clonal complexes.
A retrospective analysis of 55 clinical B. cereus isolates submitted to the Centers for Disease Control and Prevention between 1954 and 2004 was conducted. Clinical isolates from severe infections (n = 27), gastrointestinal (GI) illness (n = 18), and associated isolates from food (n = 10) were selected for analysis using MLST. The 55 isolates were diverse and comprised 38 sequence types (ST) in two distinct clades. Of the 27 isolates associated with serious illness, 13 clustered in clade 1 while 14 were in clade 2. Isolates associated with GI illness were also found throughout clades 1 and 2, while no isolates in this study belonged to clade 3. All the isolates from this study belonging to the clade 1/cereus III lineage were associated with severe disease while isolates belonging to clade1/cereus II contained isolates primarily associated with severe disease and emetic illness. Only three STs were observed more than once for epidemiologically distinct isolates.
STs of clinical B. cereus isolates were phylogenetically diverse and distributed among two of three previously described clades. Greater numbers of strains will need to be analyzed to confirm if specific lineages or clonal complexes are more likely to contain clinical isolates or be associated with specific illness, similar to B. anthracis and emetic B. cereus isolates.
Bacillus anthracis; SNP; MLVA; VNTR; Ames strain; letter
Bacillus anthracis, the etiologic agent of anthrax, has been widely described as a genetically monomorphic species. We used both multiple-locus variable-number tandem-repeat analysis (MLVA) and pagA gene sequencing to determine the genetic diversity of a historical collection of B. anthracis isolates collected from the 1950s to the 1980s from various geographic locations and sources. We sequenced the pagA gene of 124 diverse B. anthracis isolates and found all previously identified B. anthracis pagA types except type 4. Sixty-three of the 124 B. anthracis strains were identified as pagA type 6, while 44 were pagA type 5, 12 were pagA type 1, and individual isolates were identified for types 2 and 3, respectively. Two new pagA genotypes were discovered in three environmental isolates within the historical collection. Two isolates had the same new genotype, and an additional isolate produced a second new genotype. MLVA detected 22 previously described genotypes in the historical collection. In addition, 33 new MLVA genotypes were found. For 11 isolates, an MLVA genotype could not be assigned because one or more alleles did not amplify. While only two additional B. anthracis pagA types were identified, in two instances, the use of pagA sequencing discriminated isolates with the same MLVA genotype. MLVA revealed that 39 of the 124 isolates were previously undocumented genotypes and that 1 isolate was found to be in the C cluster when it was subtyped by MLVA.
Bacillus cereus is ubiquitous in nature, and while most isolates appear to be harmless, some are associated with food-borne illnesses, periodontal diseases, and other more serious infections. In one such infection, B. cereus G9241 was identified as the causative agent of a severe pneumonia in a Louisiana welder in 1994. This isolate was found to harbor most of the B. anthracis virulence plasmid pXO1 (13). Here we report the characterization of two clinical and one environmental B. cereus isolate collected during an investigation of two fatal pneumonia cases in Texas metal workers. Molecular subtyping revealed that the two cases were not caused by the same strain. However, one of the three isolates was indistinguishable from B. cereus G9241. PCR analysis demonstrated that both clinical isolates contained B. anthracis pXO1 toxin genes. One clinical isolate and the environmental isolate collected from that victim's worksite contained the cap A, B, and C genes required for capsule biosynthesis in B. anthracis. Both clinical isolates expressed a capsule; however, neither was composed of poly-d-glutamic acid. Although most B. cereus isolates are not opportunistic pathogens and only a limited number cause food-borne illnesses, these results demonstrate that some B. cereus strains can cause severe and even fatal infections in patients who appear to be otherwise healthy.
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.
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.
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.
The plasmid profiles of 619 cultures of Bacillus anthracis which had been isolated and stored between 1954 and 1989 were analyzed using the Laboratory Response Network real-time PCR assay targeting a chromosomal marker and both virulence plasmids (pXO1 and pXO2). The cultures were stored at ambient temperature on tryptic soy agar slants overlaid with mineral oil. When data were stratified by decade, there was a decreasing linear trend in the proportion of strains containing both plasmids with increased storage time (P < 0.001). There was no significant difference in the proportion of strains containing only pXO1 or strains containing only pXO2 (P = 0.25), but there was a statistical interdependence between the two plasmids (P = 0.004). Loss of viability of B. anthracis cultures stored on agar slants is also discussed.
We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.
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.
Bacillus anthracis; DFA
We determined the patterns of antimicrobial susceptibility of 65 isolates of Bacillus anthracis (50 historical and 15 recent U.S. clinical isolates) to nine antimicrobial agents using the National Committee for Clinical Laboratory Standards (NCCLS) broth microdilution reference method. The results for the 50 historical B. anthracis isolates obtained by the broth microdilution method were compared to those generated by the Etest agar gradient diffusion method. One isolate of B. anthracis was β-lactamase positive and resistant to penicillin (MIC, 128 μg/ml); a second isolate, which was β-lactamase negative, was borderline penicillin resistant, with the penicillin MICs for the isolate varying from 0.12 to 0.25 μg/ml; and the remainder of the isolates were β-lactamase negative and penicillin susceptible (MICs, ≤0.12 μg/ml). Approximately 78% of the isolates showed reduced susceptibility to ceftriaxone (MICs, ≥16 μg/ml). All B. anthracis isolates were susceptible to chloramphenicol (MICs, ≤8 μg/ml), ciprofloxacin (MICs, ≤ 1 μg/ml), clindamycin (MICs, ≤0.5 μg/ml), rifampin (MICs, ≤0.5 μg/ml), tetracycline (MICs, ≤0.06 μg/ml), and vancomycin (MICs, ≤2 μg/ml) by use of NCCLS breakpoints for staphylococci. All 15 recent B. anthracis isolates from the United States were susceptible to penicillin, doxycycline, and ciprofloxacin. By use of the susceptibility breakpoint for staphylococci of ≤0.5 μg/ml, 97% of the B. anthracis isolates tested would have been categorized as intermediate to erythromycin. No statistically significant difference was found between the results of broth microdilution testing and the results of the Etest method for any of the antimicrobial agents tested; however, the results for penicillin obtained by the Etest were 1 to 9 dilutions lower than those obtained by the broth microdilution method. The differences in the penicillin MICs by the Etest method and the difficulties of reading the Etest results through the glass of a biological safety cabinet may limit the utility of this alternate susceptibility testing method for B. anthracis isolates.
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.
Corynebacterium diphtheriae; molecular epidemiology; Russia
Molecular characterization of 53 U.S. and Canadian Corynebacterium diphtheriae isolates by multilocus enzyme electrophoresis, ribotyping, and random amplified polymorphic DNA showed that strains with distinct molecular subtypes have persisted in the United States and Canada for at least 25 years. These strains are endemic rather than imported from countries with current endemic or epidemic diphtheria.