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.

Rapid Capsular Antigen Immunoassay for Diagnosis of Inhalational Anthrax: Preclinical Studies and Evaluation in a Nonhuman Primate Model.

Inhalational anthrax is a fatal infectious disease. Rapid and effective treatment is critically dependent on early and accurate diagnosis. Blood culture followed by identification and confirmation may take days to provide clinically relevant information. In contrast, immunoassay for a shed antigen, the capsular polypeptide gamma-d-polyglutamate (γDPGA), can provide rapid results at the point of care. In this study, a lateral flow immunoassay for γDPGA was evaluated in a robust nonhuman primate model of inhalational anthrax. The results showed that the time to a positive result with the rapid test using either serum or blood as a clinical specimen was similar to the time after infection when a blood culture became positive. In vitro testing showed that the test was equally sensitive with cultures of the three major clades of Bacillus anthracis. Cultures from other Bacillus spp. that are known to produce γDPGA also produced positive results. The test was negative with human sera from 200 normal subjects and 45 subjects with culture-confirmed nonanthrax bacterial or fungal sepsis. Taken together, the results showed that immunoassay for γDPGA is an effective surrogate for blood culture in a relevant cynomolgus monkey model of inhalational anthrax. The test would be a valuable aid in early diagnosis of anthrax, which is critical for rapid intervention and a positive outcome. Use of the test could facilitate triage of patients with signs and symptoms of anthrax in a mass-exposure incident and in low-resource settings where laboratory resources are not readily available. IMPORTANCE Patient outcome in anthrax is critically dependent on early diagnosis followed by effective treatment. We describe a rapid lateral flow immunoassay that detects capsular antigen of Bacillus anthracis that is shed into blood during infection. The test was evaluated in a robust cynomolgus monkey model of inhalational anthrax. Rapid detection of capsular antigen is an effective surrogate for the time-consuming and laboratory-intensive diagnosis by blood culture, direct fluorescent antibody staining, or other molecular testing. The test can be performed at the point of patient contact, is rapid and inexpensive, and can be used by individuals with minimal training.

Case Report: A Fatal Case of Latent Melioidosis Activated by COVID-19.

Melioidosis, endemic in Southeast Asia and Northern Australia, is an uncommon but frequently fatal opportunistic infection caused by the Gram-negative saprophyte Burkholderia pseudomallei. We describe the first reported case of activation of latent melioidosis concurrent with COVID-19-associated lymphopenia and neutropenia in the setting of poorly controlled diabetes. A 43-year-old HIV-positive, diabetic man presented to the emergency department with persistent chills and progressive dyspnea. He was admitted for hypoxia. Chest X-ray showed bilateral parenchymal infiltrates suspicious for COVID-19. Shortly after admission, he became acutely encephalopathic, had a generalized seizure, and was transferred to the intensive care unit after intubation. Further workup showed severe neutropenia and lymphopenia. The patient received empiric antimicrobial coverage and was found to be severe acute respiratory syndrome coronavirus 2 positive. He deteriorated rapidly with refractory shock and persistent hypoxemia, and died 40 hours after admission. Blood cultures and sputum cultures obtained via bronchoalveolar lavage returned positive for Burkholderia pseudomallei. Given confirmed compliance with antiretrovirals, stable CD4 counts, and no recent foreign travel, the patient likely contracted the B. pseudomallei infection from travel to Southeast Asia many years prior and only became symptomatic after succumbing to severe acute respiratory syndrome coronavirus 2 infection. This case highlights the importance of considering activation of latent opportunistic infections by COVID-19 in immunocompromised patients.

Human Melioidosis Caused by Novel Transmission of Burkholderia pseudomallei from Freshwater Home Aquarium, United States1.

Nearly all cases of melioidosis in the continental United States are related to international travel to areas to which Burkholderia pseudomallei, the bacterium that causes melioidosis, is endemic. We report the diagnosis and clinical course of melioidosis in a patient from the United States who had no international travel history and the public health investigation to determine the source of exposure. We tested environmental samples collected from the patient's home for B. pseudomallei by PCR and culture. Whole-genome sequencing was conducted on PCR-positive environmental samples, and results were compared with sequences from the patient's clinical specimen. Three PCR-positive environmental samples, all collected from a freshwater home aquarium that had contained imported tropical fish, were a genetic match to the clinical isolate from the patient. This finding suggests a novel route of exposure and a potential for importation of B. pseudomallei, a select agent, into the United States from disease-endemic areas.

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.

Differentiating New from Newly Detected: Melioidosis in Yap, Federated States of Micronesia.

Melioidosis is a bacterial infection caused by exposure to water or soil that contains Burkholderia pseudomallei (Bp). Burkholderia pseudomallei is endemic to many tropical and subtropical areas of the world. In 2013, the first case of melioidosis was recognized in Yap, the Federated States of Micronesia. Six additional cases were identified in the subsequent 3 years. An investigation was initiated to understand the epidemiology of melioidosis in Yap. Serum from family and community members of the identified cases were tested for antibodies to Bp. Archived serum from a 2007 Zika serosurvey were also tested for antibodies to Bp. Sequencing of bacterial isolates was performed to understand bacterial phylogeny. Soil and water were tested for the presence of Bp in the environment by culture and PCR. None of the affected patients had a history of travel to melioidosis-endemic countries. Two of the 34 (5.8%) samples from the field investigation and 67 (11.7%) of the historical samples demonstrated serologic evidence of prior Bp exposure. No Bp were detected from 30 soil or water samples. Genotype analysis showed highly related Bp isolates that were unique to Yap. Melioidosis is likely to be endemic to Yap; however, it has only recently been recognized by the clinical community in country. Further investigation is needed to understand the local sites that harbor Bp and represent the highest risk to the community.

Anthrax Epizootic in Wildlife, Bwabwata National Park, Namibia, 2017.

In late September 2017, Bwabwata National Park in Namibia experienced a sudden die-off of hippopotamuses and Cape buffalo. A multiorganizational response was initiated, involving several ministries within Namibia and the US Centers for Disease Control and Prevention. Rapid interventions resulted in zero human or livestock cases associated with this epizootic.

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.

Bacillus anthracis gamma phage lysis among soil bacteria: an update on test specificity.

BACKGROUND: Bacillus anthracis, which causes anthrax in humans and animals, is enzootic in parts of the U.S. state of Texas where cases are typically reported in animals annually. The gamma phage lysis assay is a common diagnostic method for identification of B. anthracis and is based on the bacterium's susceptibility to lysis. This test has been shown to be 97% specific for B. anthracis, as a small number of strains of other Bacillus spp. are known to be susceptible. In this study, we evaluated the performance of a combination of B. anthracis diagnostic assays on 700 aerobic, spore-forming isolates recovered from soil collected in Texas. These assays include phenotypic descriptions, gamma phage susceptibility, and real-time polymerase chain reaction specific for B. anthracis. Gamma phage-susceptible isolates were also tested using cell wall and capsule direct fluorescent-antibody assays specific for B. anthracis. Gamma phage-susceptible isolates that were ruled out as B. anthracis were identified by 16S rRNA gene sequencing. FINDINGS: We identified 29 gamma phage-susceptible isolates. One was confirmed as B. anthracis, while the other 28 isolates were ruled out for B. anthracis by the other diagnostic tests. Using 16S rRNA gene sequencing results, we identified these isolates as members of the B. cereus group, Bacillus sp. (not within B. cereus group), Lysinibacillus spp., and Solibacillus silvestris. Based on these results, we report a specificity of 96% for gamma phage lysis as a diagnostic test for B. anthracis, and identified susceptible isolates outside the Bacillus genus. CONCLUSIONS: In this study we found gamma phage susceptibility to be consistent with previously reported results. However, we identified non-B. anthracis environmental isolates (including isolates from genera other than Bacillus) that are susceptible to gamma phage lysis. To date, susceptibility to gamma phage lysis has not been reported in genera other than Bacillus. Though these isolates are not of clinical origin, description of unexpected positives is important, especially as new diagnostic assays for B. anthracis are being developed based on gamma phage lysis or gamma phage proteins.

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.