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Diagnostic challenges within the Bacillus cereus-group: finding the beast without teeth

The Bacillus cereus-group (B. cereus sensu lato) includes common, usually avirulent species, often considered contaminants of patient samples in routine microbiological diagnostics, as well as the highly virulent B. anthracis. Here we describe 16 isolates from 15 patients, identified as B. cereus-group using a MALDI-TOF MS standard database. Whole genome sequencing (WGS) analysis identified five of the isolates as B. anthracis species not carrying the typical virulence plasmids pXO1 and pXO2, four isolates as B. paranthracis, three as B. cereus sensu stricto, two as B. thuringiensis, one as B. mobilis, and one isolate represents a previously undefined species of Bacillus (B. basilensis sp. nov.). More detailed analysis using alternative MALDI-TOF MS databases, biochemical phenotyping, and diagnostic PCRs, gave further conflicting species results. These cases highlight the difficulties in identifying avirulent B. anthracis within the B. cereus-group using standard methods. WGS and alternative MALDI-TOF MS databases offer more accurate species identification, but so far are not routinely applied. We discuss the diagnostic resolution and discrepancies of various identification methods.

virulence of B. anthracis is enabled by two virulence plasmids, pXO1 and pXO2, carrying genes encoding for the tripartite toxin and the poly-γ-D-glutamic acid capsule, respectively [5,6].
In clinical routine diagnostics, the fast and reliable identification of B. cereus and B. anthracis is crucial because of the pathogenic and biothreat potential of B. anthracis. The identification is classically based on morphological features. B. anthracis is typically described as non-hemolytic on blood agar with a "Medusa's head" colony morphology. In contrast, B. cereus causes hemolysis and has a grey matt colony morphology [7,8]. Additionally, they can be distinguished by motility, and penicillin and phage γ susceptibility, where B. anthracis shows susceptibility to both [7][8][9][10][11]. Biochemical reaction profiles can differentiate B. anthracis from other members of the B. cereus-group [10,12]. However, phenotypic characteristics may vary within and among species resulting in inconsistent species identification [10]. Species distinction by 16S ribosomal RNA gene sequencing has proven to be difficult [3,5,13]. Standard MALDI-TOF mass spectrometry (MALDI-TOF MS) is faster but reliable differentiation within the B. cereus-group is difficult using commercial databases [14,15]. Alternative diagnostic approaches include specific PCRs targeting toxins, virulence plasmids and chromosomal markers [16]. In order to investigate the ancestral relatedness of isolates and assign species accordingly, genomics provides the gold standard. Many methods have been used to classify genomes within the B. cereus-group: digital DNA:DNA hybridization (dDDH) [3]; the Genome Taxonomy Database (GTDB) [10,17], and average nucleotide identity (ANI), perhaps with a proposed genomospecies cutoff at 92.5% rather than the accepted 95-96% cutoff [18]. Using any of these methods, cases of discrepancy in allocating isolates to species have been described. To add to the confusion, the presence of virulence factors does not always correlate with species identification, as some isolates closely related to B. anthracis do not carry pXO1 and pXO2 [3,19,20]. Conversely, these plasmids have been found within the genome of other Bacillus species [21]. In this study we analyze 16 clinical B. cereus-group isolates from 15 patients, assigned to the B. cereus-group by clinical and morphological features and the MALDI-TOF MS Bruker standard database (sDB). We describe the isolates in depth using phenotypic and genomic methods to highlight the diagnostic complexity.
2Material and methods 2.1Sample collection and culture The 16 B. cereus-group isolates ( Table 1) were collected during July 2019, and between July and September 2021, as well as isolates from interesting clinical cases across this timeframe. Isolates were cultured on Columbia blood agar, supplemented with 5% sheep blood (BD Becton Dickinson, New Jersey, USA) at 37°C under aerobic atmosphere supplemented with 5% CO 2 over 24 hours.

2.2Evaluation of clinical relevance
All clinical cases were evaluated regarding the clinical relevance of the B. cereus-group isolates by an infectious diseases specialist.
Antibiotic susceptibility testing (AST) for penicillin, clindamycin, levofloxacin and vancomycin was performed using the gradient diffusion method (MIC Test Strip, Liofilchem®, Italy) on Müller-Hinton agar (BD Becton Dickinson, New Jersey, USA) resulting in minimum inhibitory concentrations (MIC) in mg/L. Penicillin was interpreted by Clinical and Laboratory Standards Institute (CLSI) breakpoints, and clindamycin, levofloxacin and vancomycin by European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints [25,26]. Specific PCR assays for B. anthracis targeted the virulence plasmids pXO1 and pXO2 (Supplemental material Table 2), and the chromosomal gyrA gene [27].

Ethical statement
This retrospective observational case-series has been approved by the local ethical committee following the standards of the Swiss Human Research Act (EKNZ 2021-02112). Whenever possible written informed consent was collected, no patient refused participation.  Table 1. Of the 16 isolates, six isolates were classified as clinically relevant, while 10 were classified as not relevant. Routine analysis using the MALDI-TOF MS sDB assigned 14 isolates to B. cereus and two to B. thuringiensis, with high scores (>2.0) ( Table 2).
3.1.1Whole genome sequencing and species identification. All 16 isolates underwent genome sequencing for further characterization (Table 3).
The genome assemblies were used to identify the isolates using several methods (Table 4). GTDB and TYGS give more species-level results, and agree more than the other methods, providing the striking finding that five genomes are defined as belonging to B. anthracis (Fig. 1). This species was not reported at all by 16S rRNA gene identification or rMLST. ANI comparisons for these genomes showed a majority of top hits (96-99.8%) to genomes assigned to B. anthracis (data not shown). None of the isolates were found to contain complete plasmids pXO1, pXO2, or alternative capsule-carrying plasmid pBC218, with all isolates mapping to only 5-53% of these references. None were found to contain anthrax toxins nor capsule genes cya, lef, pagA or capABCDE. All genomes carry the  nov., all isolates carry bla2 BcII family beta-lactamase (A7J11_00039, A7J11_05308 or A7J11_05161) at >91% identity, although this gene was disrupted in isolate 608121-20. These genes can lead to penicillin resistance when expression levels are high [34]. These results provide evidence that five isolates are most closely related to B. anthracis, which was not seen from the routine microbiology. Given these discrepant results, and the lack of expert consensus on species identification, further identification methods were performed.

3.2Identification based on phenotype
All 16 isolates showed the classical morphological characteristics of B. cereus: grey, matt colonies exhibiting large double-zone β-hemolysis. A representative example is shown in Supplemental material Fig. 1. Using the VITEK®2 phenotypic test, all isolates were assigned to B. cereus/thuringiensis/mycoides with an identification probability ranging from 89% to 99%, which is the highest level of resolution provided by VITEK®2 BCL card (manufacturer's information) ( Table 2). For the four key reactions identified, no isolate showed a similar profile to the reference B. anthracis, and all isolates showed a positive tyrosine arylamidase reaction (Supplemental material Table 3 for key reactions and Supplemental material Table 4 for more detailed reaction profiles). AST showed that all isolates were resistant to penicillin, but susceptible to clindamycin and vancomycin, and susceptible to increased exposure with levofloxacin (Supplemental material Table 5).

3.3Identification based on MALDI-TOF MS and virulence specific real-time PCR
Through re-measuring the MALDI-TOF MS spectra of the isolates and using the SR DB, we assigned six isolates to B. anthracis with high scores (Table 2). Using the Mabritec® PAPMID™ BCG-Classifier database, five isolates were identified as B. anthracis without pXO1 and pXO2, four as B. paranthracis, three as B. cereus, two as B. thuringiensis, one as B. mobilis and one as a previously undescribed "genomospecies 13" described here as B. basilensis sp. nov. (Table 2). B. anthracis specific real-time PCRs were performed on all 16 isolates. All were negative for pXO1, pXO2, and gyrA, this latter likely due can be seen to belong to the species B. anthracis, using this standard cut-off (Table 4). 6 New Microbes and New Infections, Volume 49-50 Number C, ---2022 to a C-T mutation in the assay probe binding site in the B. anthracis isolates (Supplementary material Fig. 2).

4Discussion
Isolate identification within the B. cereus-group is challenging. Of the various phenotypic and genotypic approaches used in this study to characterize the 16 isolates, we consider genomic analysis by TYGS with appropriate additional comparators to provide the most accurate species assignation. The TYGS results were in agreement with those from Mabritec® PAPMID™ BCG-Classifier database in all cases, and assigned five isolates to B. anthracis, albeit without virulence plasmids, four isolates to B. paranthracis, three to B. cereus, two to B. thuringiensis, one to B. mobilis, and one isolate was identified as a new species, B. basilensis sp. nov. This shows the species diversity that can be hidden behind a generic diagnosis of "B. cereus-group".
Illustrating the lack of species distinction in morphological and phenotypic tests, all our isolates showed morphology and AST patterns typical for B. cereus. The biochemical reaction profile of all our isolates was consistent with B. cereus/thuringiensis/mycoides, showing that the VITEK®2 BCL card offered no additional diagnostic support. Only 5/16 (31.3%) isolates were correctly identified at the species level using routine microbiological methods. Of note, B. paranthracis and B. mobilis cannot be identified by VITEK®2 BCL card.
The original definition of B. anthracis was tightly linked to the presence of the anthrax toxin, making the definition of B. anthracis particularly problematic. Many remain convinced that only tightly related, monomorphic, toxin-carrying strains qualify for the name [18,33]. By dDDH analysis, using the 70% cutoff, the species can be more broadly defined, including nontoxin carrying strains, and those described here [3,19]. B. anthracis is absent from the TYGS database, as no type strain genome sequence exists, which complicates identification, and shows that these tools must be used with caution. B. anthracis exists in the rMLST database, but the isolates presented here are sufficiently divergent not to be recognized as B. anthracis by this method.
Of the isolates, 6/16 were defined as clinically relevant, of which three were identified as B. anthracis. Illumina-based WGS offers high resolution identification, but a turn-around time of several days is too slow to generate an impact in clinical management. Using the MALDI-TOF MS sDB, only 4/16 (25%) isolates were correctly identified. This is expected, since B. anthracis, B. paranthracis and B. mobilis are not in the database, and it is known that commercial standard databases do not reliably differentiate species within the B. cereus-group [15]. Even using the SR DB, only 9/16 (56,3%) isolates were correctly identified, including five correctly identified B. anthracis, but also one falsely identified as B. anthracis. Alternative methods including real-time nanopore sequencing technologies or improved MALDI-TOF MS databases would be valuable additions in these cases. The Mabritec® PAPMID™ database, an online accessible (www.mabriteccentral.com) proprietary database which supports Bruker Biotyper and VITEK® MS bioMérieux data files, is based on in silico predicted ribosomal marker peaks und uses the PAPMID™ algorithm for classification. A sophisticated sample preparation, including the use of sinapinic acid as matrix, was used and all our isolates were correctly identified to species level. The BCG-classifier used is based on deduced reference marker masses from 2204 public B. cereus-group genomes, covering 20 accepted species and 21 genomospecies.
Further improvements to MALDI-TOF MS analyses include identifying specific B. anthracis biomarker candidates [15], using software such as ClinProTools (Bruker) and Matlab (The Mathworks Inc.), combined with different packages for use in R [14,35], using different matrices [35], and direct detection of anthrax toxin component, the lethal factor [36]. Comprehensive databases can increase the probability of confirmed identification up to 93% [37]. However, these methods need to be evaluated for use in routine diagnostics.
The implications of species mis-identification need to be considered. Defining genomic ancestry does not help in assessment of virulence potential, which is of most clinical and biowarfare agent concern. To address this, suggestions have been made for a new taxonomic nomenclature for the B. cereus-group that takes into account phenotypic and genomic characteristics, defining a biovar concept [18] or using BTyper [38]. Anthrax is a notifiable disease: it remains to be clarified whether a non-virulent isolate belonging to B. anthracis requires urgent notification.
Using standard genomic methods of species identification, we have shown that isolates belonging to B. anthracis are frequently found in the clinical routine laboratory, albeit nontoxin-carrying isolates representing more diverse members of the species. We find that routine morphological, phenotypic, and standard MALDI-TOF MS methods are insufficient for accurate species identification within the B. cereus-group, and additional methods should be applied in difficult cases.

4.1Taxonomic description of Bacillus basilensis sp. nov.
A Gram-positive, rod-shaped bacillus (403507-21) was isolated from human superficial skin swab. On the basis of MALDI-TOF MS identification, the isolate was assigned to the Bacillus cereusgroup. 16S rRNA gene sequence (accession number: OV062800) analysis shows over 99.8% nucleotide identity to isolates belonging to species Bacillus proteolyticus, Bacillus