Retrospective Screening of Anthrax-like Disease Induced by Bacillus tropicus str. JMT from Chinese Soft-Shell Turtles in Taiwan

Abstract

Bacillus cereus is ubiquitous in the environment and a well-known causative agent of foodborne disease. Surprisingly, more and more emerging strains of atypical B. cereus have been identified and related to severe disease in humans and mammals such as chimpanzees, apes, and bovine. Recently, the atypical B. cereus isolates, which mainly derive from North America and Africa, have drawn great attention due to the potential risk of zoonosis. The cluster of B. cereus carries several anthrax-like virulent genes that are implicated in lethal disease. However, in non-mammals, the distribution of atypical B. cereus is still unknown. In this study, we conducted a retrospective screening of the 32 isolates of Bacillus spp. from diseased Chinese soft-shelled turtles from 2016 to 2020. To recognize the causative agent, we used various methods, such as sequencing analysis using PCR-amplification of the 16S rRNA gene, multiplex PCR for discriminating, and colony morphology by following previous studies. Furthermore, the digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values were calculated, respectively, below the 70 and 96% cutoff to define species boundaries. According to the summarized results, the pathogen is taxonomically classified as Bacillus tropicus str. JMT (previous atypical Bacillus cereus). Subsequently, analyses such as targeting the unique genes using PCR and visual observation of the bacteria under various staining techniques were implemented in our study. Our findings show that all (32/32, 100%) isolates in this retrospective screening share similar phenotypical properties and carry the protective antigen (PA), edema factor (EF), hyaluronic acid (HA), and exopolysaccharide (Bps) genes on their plasmids. In this study, the results indicate that the geographic distribution and host range of B. tropicus were previously underestimated.

Get to Know Your Neighbors: Characterization of Close Bacillus anthracis Isolates and Toxin Profile Diversity in the Bacillus cereus Group

“…B. anthracis biovar Anthracis (or B. Anthracis) has a long history as a life-threatening infectious agent to humans and animals worldwide [66] and has been extensively studied since the anthrax letter events in 2001 and the subsequent anthrax outbreaks [67,68]. The production of anthrax toxin has long been considered restricted to the B. anthracis species, but anthrax-causing strains have been characterized since 2006 outside the B. anthracis lineage in humans [8,23], great apes [24], in a kangaroo [8] and, recently, in a soft-shell turtle [26]. These B. mosaicus biovar Anthracis strains, previously referred to as "anthrax-like" strains, may exhibit different capsular composition [20] and are so far described as close B. anthracis neighbors or are related to the B. tropicus species [36] (see Figure 1 and Table 1).…”

“…Recently, a retrospective screening of an anthrax-like disease induced by a strain of Bacillus tropicus from Chinese turtles in Taiwan reinforced the idea that the host range and geographic distribution of atypical B. cereus s.l. are by far underestimated [26].…”