Investigating Anthrax-Associated Virulence Genes among Archival and Contemporary Bacillus cereus Group Genomes.

Bacillus anthracis causes anthrax through virulence factors encoded on two plasmids. However, non-B. anthracis organisms within the closely related, environmentally ubiquitous Bacillus cereus group (BCG) may cause an anthrax-like disease in humans through the partial adoption of anthrax-associated virulence genes, challenging the definition of anthrax disease. To elucidate these phenomena and their evolutionary past, we performed whole-genome sequencing on non-anthracis BCG isolates, including 93 archival (1967-2003) and 5 contemporary isolates (2019-2023). We produced annotated genomic assemblies and performed a pan-genome analysis to identify evidence of virulence gene homology and virulence gene acquisition by linear inheritance or horizontal gene transfer. At least one anthrax-associated virulence gene was annotated in ten isolates. Most homologous sequences in archival isolates showed evidence of pseudogenization and subsequent gene loss. The presence or absence of accessory genes, including anthrax-associated virulence genes, aligned with the phylogenetic structure of the BCG core genome. These findings support the hypothesis that anthrax-associated virulence genes were inherited from a common ancestor in the BCG and were retained or lost across different lineages, and contribute to a growing body of work informing public health strategies related to anthrax surveillance and identification.

Strains Associated with Two 2020 Welder Anthrax Cases in the United States Belong to Separate Lineages within Bacillus cereus sensu lato.

Anthrax-causing members of Bacillus cereus sensu lato (s.l.) pose a serious threat to public health. While most anthrax-causing strains resemble B. anthracis phenotypically, rare cases of anthrax-like illness caused by strains resembling "B. cereus" have been reported. Here, whole-genome sequencing was used to characterize three B. cereus s.l. isolates associated with two 2020 welder anthrax cases in the United States, which resembled "B. cereus" phenotypically. Comparison of the three genomes sequenced here to all publicly available, high-quality B. cereus s.l. genomes (n = 2890 total genomes) demonstrated that genomes associated with each case effectively belonged to separate species at the conventional 95% average nucleotide identity prokaryotic species threshold. Two PubMLST sequence type 78 (ST78) genomes affiliated with a case in Louisiana were most closely related to B. tropicus and possessed genes encoding the Bps exopolysaccharide capsule, as well as hemolysin BL (Hbl) and cytotoxin K (CytK). Comparatively, a ST108 genome associated with a case in Texas was most closely related to B. anthracis; however, like other anthrax-causing strains most closely related to B. anthracis, this genome did not possess Bps-, Hbl-, or CytK-encoding genes. Overall, results presented here provide insights into the evolution of anthrax-causing B. cereus s.l.

Outbreak of cutaneous anthrax associated with handling meat of dead cows in Southwestern Uganda, May 2018.

BACKGROUND: Anthrax is a zoonotic infection caused by the bacteria Bacillus anthracis. Humans acquire cutaneous infection through contact with infected animals or animal products. On May 6, 2018, three cows suddenly died on a farm in Kiruhura District. Shortly afterwards, a sub-county chief in Kiruhura District received reports of humans with suspected cutaneous anthrax in the same district. The patients had reportedly participated in the butchery and consumption of meat from the dead cows. We investigated to determine the magnitude of the outbreak, identify exposures associated with illness, and suggest evidence-based control measures. METHODS: We conducted a retrospective cohort study among persons whose households received any of the cow meat. We defined a suspected human cutaneous anthrax case as new skin lesions (e.g., papule, vesicle, or eschar) in a resident of Kiruhura District from 1 to 26 May 2018. A confirmed case was a suspected case with a lesion testing positive for B. anthracis by polymerase chain reaction (PCR). We identified cases through medical record review at Engari Health Centre and active case finding in the community. RESULTS: Of the 95 persons in the cohort, 22 were case-patients (2 confirmed and 20 suspected, 0 fatal cases) and 73 were non-case household members. The epidemic curve indicated multiple point-source exposures starting on May 6, when the dead cows were butchered. Among households receiving cow meat, participating in slaughtering (RR = 5.3, 95% CI 3.2-8.3), skinning (RR = 4.7, 95% CI = 3.1-7.0), cleaning waste (RR = 4.5, 95% CI = 3.1-6.6), and carrying meat (RR = 3.9, 95% CI = 2.2-7.1) increased the risk of infection. CONCLUSIONS: This cutaneous anthrax outbreak was caused by handling infected animal carcasses. We suggested to the Ministry of Agriculture, Animal Industry and Fisheries to strengthen surveillance for possible veterinary anthrax and ensure that communities do not consume carcasses of livestock that died suddenly. We also suggested that the Ministry of Health equip health facilities with first-line antibiotics for community members during outbreaks.

Epidemiologic Investigation of Two Welder's Anthrax Cases Caused by Bacillus Cereus Group Bacteria: Occupational Link Established by Environmental Detection.

Abstract Bacillus cereus group bacteria containing the anthrax toxin genes can cause fatal anthrax pneumonia in welders. Two welder's anthrax cases identified in 2020 were investigated to determine the source of each patient's exposure. Environmental sampling was performed at locations where each patient had recent exposure to soil and dust. Samples were tested for the anthrax toxin genes by real-time PCR, and culture was performed on positive samples to identify whether any environmental isolates matched the patient's clinical isolate. A total of 185 environmental samples were collected in investigation A for patient A and 108 samples in investigation B for patient B. All samples from investigation B were real-time PCR-negative, but 14 (8%) samples from investigation A were positive, including 10 from patient A's worksite and 4 from his work-related clothing and gear. An isolate genetically matching the one recovered from patient A was successfully cultured from a worksite soil sample. All welder's anthrax cases should be investigated to determine the source of exposure, which may be linked to their worksite. Welding and metalworking employers should consider conducting a workplace hazard assessment and implementing controls to reduce the risk of occupationally associated illnesses including welder's anthrax.

Welder's Anthrax: A Review of an Occupational Disease.

Since 1997, nine cases of severe pneumonia, caused by species within the B. cereus group and with a presentation similar to that of inhalation anthrax, were reported in seemingly immunocompetent metalworkers, with most being welders. In seven of the cases, isolates were found to harbor a plasmid similar to the B. anthracis pXO1 that encodes anthrax toxins. In this paper, we review the literature on the B. cereus group spp. pneumonia among welders and other metalworkers, which we term welder's anthrax. We describe the epidemiology, including more information on two cases of welder's anthrax in 2020. We also describe the health risks associated with welding, potential mechanisms of infection and pathological damage, prevention measures according to the hierarchy of controls, and clinical and public health considerations. Considering occupational risk factors and controlling exposure to welding fumes and gases among workers, according to the hierarchy of controls, should help prevent disease transmission in the workplace.

Ecological Niche Model of Bacillus cereus Group Isolates Containing a Homologue of the pXO1 Anthrax Toxin Genes Infecting Metalworkers in the United States.

While Bacillus cereus typically causes opportunistic infections in humans, within the last three decades, severe and fatal infections caused by isolates of the B. cereus group harboring anthrax toxin genes have been reported in the United States. From 1994 to 2020, seven cases of anthrax-like illness resulting from these isolates have been identified. With one exception, the cases have occurred in the Gulf States region of the United States among metalworkers. We aimed to develop an ecological niche model (ENM) to estimate a spatial area conducive to the survival of these organisms based on the presence of known human infections and environmental variables. The estimated ecological niche for B. cereus was modeled with the maximum entropy algorithm (Maxent). Environmental variables contributing most to the model were soil characteristics (cation exchange capacity, carbon content, soil pH), temperature, enhanced vegetation index (EVI), and land surface temperature (LST). Much of the suitable environments were located throughout the Gulf Coast Plain, Texas Backland Prairies, East Central Texas Plains, Edwards Plateau, Cross Timbers, Mississippi Alluvial Plain, and Central Great Plains. These findings may provide additional guidance to narrow potential risk areas to efficiently communicate messages to metalworkers and potentially identify individuals who may benefit from the anthrax vaccine.

Melioidosis in Cynomolgus Macaques ( Macaca Fascicularis ) Imported to the United States from Cambodia.

Melioidosis, a potentially fatal infectious disease of humans and animals, including nonhuman primates (NHPs), is caused by the high-consequence pathogen Burkholderia pseudomallei. This environmental bacterium is found in the soil and water of tropical regions, such as Southeast Asia, where melioidosis is endemic. The global movement of humans and animals can introduce B. pseudomallei into nonendemic regions of the United States, where environmental conditions could allow establishment of the organism. Approximately 60% of NHPs imported into the United States originate in countries considered endemic for melioidosis. To prevent the introduction of infectious agents to the United States, the Centers for Disease Control and Prevention (CDC) requires newly imported NHPs to be quarantined for at least 31 d, during which time their health is closely monitored. Most diseases of public health concern that are transmissible from imported NHPs have relatively short incubation periods that fall within the 31-d quarantine period. However, animals infected with B. pseudomallei may appear healthy for months to years before showing signs of illness, during which time they can shed the organism into the environment. Melioidosis presents diagnostic challenges because it causes nonspecific clinical signs, serologic screening can produce unreliable results, and culture isolates are often misidentified on rapid commercial testing systems. Here, we present a case of melioidosis in a cynomolgus macaque (Macaca fascicularis) that developed a subcutaneous abscess after importation from Cambodia to the United States. The bacterial isolate from the abscess was initially misidentified on a commercial test. This case emphasizes the possibility of melioidosis in NHPs imported from endemic countries and its associated diagnostic challenges. If melioidosis is suspected, diagnostic samples and culture isolates should be submitted to a laboratory in the CDC Laboratory Response Network for conclusive identification and characterization of the pathogen.

Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness.

Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response.

Genomic Characterization and Copy Number Variation of Bacillus anthracis Plasmids pXO1 and pXO2 in a Historical Collection of 412 Strains.

Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. However, the extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of the Bacillus cereus sensu lato group remain unclear. To gain new insights into these issues, we sequenced 412 B. anthracis strains representing the total phylogenetic and ecological diversity of the species. Our results revealed that B. anthracis genomes carried, on average, 3.86 and 2.29 copies of pXO1 and pXO2, respectively, and also revealed a positive linear correlation between the copy numbers of pXO1 and pXO2. No correlation between the plasmid copy number and the phylogenetic relatedness of the strains was observed. However, genomes of strains isolated from animal tissues generally maintained a higher plasmid copy number than genomes of strains from environmental sources (P < 0.05 [Welch two-sample t test]). Comparisons against B. cereus genomes carrying complete or partial pXO1-like and pXO2-like plasmids showed that the plasmid-based phylogeny recapitulated that of the main chromosome, indicating limited plasmid horizontal transfer between or within these species. Comparisons of gene content revealed a closed pXO1 and pXO2 pangenome; e.g., plasmids encode <8 unique genes, on average, and a single large fragment deletion of pXO1 in one B. anthracis strain (2000031682) was detected. Collectively, our results provide a more complete view of the genomic diversity of B. anthracis plasmids, their copy number variation, and the virulence potential of other Bacillus species carrying pXO1/pXO2-like plasmids. IMPORTANCE Bacillus anthracis microorganisms are of historical and epidemiological importance and are among the most homogenous bacterial groups known, even though the B. anthracis genome is rich in mobile elements. Mobile elements can trigger the diversification of lineages; therefore, characterizing the extent of genomic variation in a large collection of strains is critical for a complete understanding of the diversity and evolution of the species. Here, we sequenced a large collection of B. anthracis strains (>400) that were recovered from human, animal, and environmental sources around the world. Our results confirmed the remarkable stability of gene content and synteny of the anthrax plasmids and revealed no signal of plasmid exchange between B. anthracis and pathogenic B. cereus isolates but rather predominantly vertical descent. These findings advance our understanding of the biology and pathogenomic evolution of B. anthracis and its plasmids.

Enhancing Surveillance and Diagnostics in Anthrax-Endemic Countries.

Naturally occurring anthrax disproportionately affects the health and economic welfare of poor, rural communities in anthrax-endemic countries. However, many of these countries have limited anthrax prevention and control programs. Effective prevention of anthrax outbreaks among humans is accomplished through routine livestock vaccination programs and prompt response to animal outbreaks. The Centers for Disease Control and Prevention uses a 2-phase framework when providing technical assistance to partners in anthrax-endemic countries. The first phase assesses and identifies areas for improvement in existing human and animal surveillance, laboratory diagnostics, and outbreak response. The second phase provides steps to implement improvements to these areas. We describe examples of implementing this framework in anthrax-endemic countries. These activities are at varying stages of completion; however, the public health impact of these initiatives has been encouraging. The anthrax framework can be extended to other zoonotic diseases to build on these efforts, improve human and animal health, and enhance global health security.

Draft Genome Sequence of Bacillus cereus LA2007, a Human-Pathogenic Isolate Harboring Anthrax-Like Plasmids.

We present the genome sequence of Bacillus cereus LA2007, a strain isolated in 2007 from a fatal pneumonia case in Louisiana. Sequence-based genome analysis revealed that LA2007 carries a plasmid highly similar to Bacillus anthracis pXO1, including the genes responsible for the production and regulation of anthrax toxin.

Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar.

Bacillus cereus isolates have been described harboring Bacillus anthracis toxin genes, most notably B. cereus G9241, and capable of causing severe and fatal pneumonias. This report describes the characterization of a B. cereus isolate, BcFL2013, associated with a naturally occurring cutaneous lesion resembling an anthrax eschar. Similar to G9241, BcFL2013 is positive for the B. anthracis pXO1 toxin genes, has a multi-locus sequence type of 78, and a pagA sequence type of 9. Whole genome sequencing confirms the similarity to G9241. In addition to the chromosome having an average nucleotide identity of 99.98% when compared to G9241, BcFL2013 harbors three plasmids with varying homology to the G9241 plasmids (pBCXO1, pBC210 and pBFH_1). This is also the first report to include serologic testing of patient specimens associated with this type of B. cereus infection which resulted in the detection of anthrax lethal factor toxemia, a quantifiable serum antibody response to protective antigen (PA), and lethal toxin neutralization activity.

Whole-genome sequencing investigation of animal-skin-drum-associated UK anthrax cases reveals evidence of mixed populations and relatedness to a US case.

There have been two anthrax cases affecting people that played and/or made animal-skin drums in the UK during the last 10 years, with single fatal occurrences in Scotland in 2006 and London in 2008. Investigations by the Health Protection Agency (now Public Health England) employing multi-locus-variable number tandem repeat analysis had previously linked the clinical cases to spores associated with animal skins and drums the patients had been in contact with. In this study, whole-genome sequencing of 23 Bacillus anthracis isolates harvested during the investigations was performed. High-quality draft assemblies of these genomes provided greater characterization of the B. anthracis strains present and placed them all upon a new branch of the global phylogeny. Although closely related, the clinical isolates from the two events, and another isolated from a drum-skin-associated case in New York in 2006, were distinct from each other. Multiple distinct genotypes were found during both investigations, implying either multiple contamination events or a single heterogeneous contamination. One environmental isolate from the Scottish incident was more closely related to London isolates than to the other Scottish isolates. As B. anthracis of this subgroup was present at both geographically and temporally distinct events, it may be more widespread at the source of contamination. All isolates were distinct from currently characterized West African strains, despite this being the likely origin of the drums and hides, therefore adding to our knowledge of B. anthracis diversity in the region.

Investigation and control of anthrax outbreak at the human-animal interface, Bhutan, 2010.

In 2010, we investigated anthrax outbreak in Bhutan. A total of 43 domestic animals died, and cutaneous anthrax developed in 9 persons, and 1 died. All affected persons had contact with the carcasses of infected animals. Comprehensive preparedness and response guidelines are needed to increase public awareness of anthrax in Bhutan.

Draft Genome Sequence of Bacillus cereus Strain BcFL2013, a Clinical Isolate Similar to G9241.

Bacillus cereus strains, such as G9241, causing anthrax-like illnesses have recently been discovered. We report the genome sequence of a clinical strain, B. cereus BcFL2013, which is similar to G9241, recovered from a patient in Florida.

Swab protocol for rapid laboratory diagnosis of cutaneous anthrax.

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.

Molecular epidemiology of anthrax cases associated with recreational use of animal hides and yarn in the United States.

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.

Lethal factor toxemia and anti-protective antigen antibody activity in naturally acquired cutaneous anthrax.

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.