Rapidly fatal infection with Bacillus cereus/thuringiensis: Genome assembly of the responsible pathogen and consideration of possibly contributing toxins

1 Summary

Bloodstream infection with Bacillus cereus/thuringiensis can be life threatening, particularly in patients who are severely immunocompromised. In this report we describe a case that progressed from asymptomatic to fatal over approximately 5 hours despite extensive resuscitation efforts. We identify the pathogen and assemble its genome, in which we find genes for toxins that may have contributed to the precipitous demise. In the context of this and other cases we discuss the possible indication for rapid appropriate antibiotic administration and potentially antitoxin treatment or toxin removal in fulminant illness in immunocompromised patients.

2. History

A previously healthy 5 year old female presented with neck and facial swelling, gum bleeding and petechiae. She was found to have leukocytosis (white blood cell count 56.7 K/cu mm, reference range: 6.0 – 17.0 K/cu mm), anemia (hemoglobin 3.4 g/dL, reference range: 11.6 – 13.6 g/dL), and thrombocytopenia (platelet count 4 K/cu mm, reference range: 150 – 350 K/cu mm). Flow cytometry on peripheral blood revealed a population of blasts comprising 90% of cellularity with a phenotype consistent with precursor B-cell acute lymphoblastic leukemia (pre-B ALL). She was started on induction chemotherapy and empiric intravenous cefepime for a febrile episode during blood transfusion. Her hospital course was otherwise stable and cefepime was discontinued on hospital day 13 with plans for discharge and administration of chemotherapy as an outpatient if afebrile after 48 hours.

Approximately 18 hours after her last cefepime dose, she began to experience abdominal discomfort though she had stable vital signs (temperature 37.9 C) and physical examination. Routine labs drawn approximately 30 minutes prior showed newly elevated transaminases (aspartate amino transferase 256 U/L, reference range: 0 – 31 U/L; alanine amino transferase 194 U/L, reference range: 0 – 31 U/L). Approximately two hours from her initial abdominal discomfort, she became febrile to 38.6 C. Within thirty minutes of becoming febrile she acutely decompensated with alterations in mental status, a dusky appearance, and worsening vital signs including tachycardia (heart rate 200 beats per minute), tachypnea (respirations 54 breaths per minute), hypoxia (oxygen saturation 64% on room air), and hypotension (blood pressure 70/43 mm Hg). Blood cultures were drawn and she received a bolus of normal saline and intravenous cefepime. Pediatric advanced life support was initiated for unstable bradycardia with progression to pulseless electrical activity. Chest radiograph showed bilateral hazy opacities with new bilateral pleural effusions. Extracorporeal membrane oxygenation was initiated. Her abdomen became increasingly rigid and distended, raising concern for abdominal compartment syndrome. Bedside decompressive laparotomy was performed revealing an engorged liver without hemoperitoneum. After approximately three hours of cardiopulmonary resuscitation, the decision was made to withdraw resuscitation efforts.

Autopsy was performed at an interval of approximately 8 hours. Externally there was edema and the skin showed scattered ecchymoses and petechiae. The thoracic cavity showed bilateral pleural effusions, pneumomediastinum, and a pericardial effusion. The lungs were approximately twice normal weight and showed patchy gray-brown discoloration. The small intestine progressed from a grossly unremarkable appearance proximally to wall hemorrhage and ischemic changes distally. The large intestine had patchy hemorrhage with extensive pneumatosis intestinalis in the transverse colon. The liver was enlarged with mottling, discoloration, and firm areas. There was no thrombus in the inferior vena cava, hepatic vein, or portal vein. There was scattered soft tissue and organ hemorrhage involving the mediastinum, diaphragm, heart, mesentery, pancreas, right kidney, adrenal glands, bladder, ovaries, and brain.

On microscopic examination, bacilli were present in the trachea, bronchi, both lungs, grossly abnormal portions of small intestine, liver, pancreas, soft tissue adjacent to the thyroid, splenic red pulp, and intracranial blood vessels. The lungs also showed vascular congestion, patchy hemorrhage, and proteinaceous debris filling the alveoli. The bacilli in the liver filled sinusoids in foci, and surrounding hepatocytes showed necrosis. The scattered bacilli in the pancreas were surrounded by acinar cell necrosis. The kidneys showed diffuse acute tubular injury, consistent with prolonged hypotension. Despite the extensive involvement of multiple organs with bacilli, there was no associated acute or chronic inflammation, consistent with the patient’s neutropenic state and likely inability to mount an immune response. Figure 1 demonstrates key microscopic findings.

Figure 1: Histology demonstrates bacilli but no inflammatory response.

Top: Abundant organisms in hepatic sinusoids. Original magnification 160x. Middle: Bacilli in fibrin thrombi in leptomeningeal vessel, brain. Original magnification 260x. Bottom: Bacilli in alveolar membranes of lung. In these areas as well as all other areas with bacilli examined, no cellular immune response was present. Original magnification 260x. All panels hematoxylin and eosin stain.

3. Identification of the pathogen, assembly of its genome, and identification of toxin genes present

Blood cultures

Two Bacillus species were recovered from blood cultures drawn at the same time during the resuscitation effort from two separate sites. Both blood bottles (BACTEC-FX, Becton Dickinson, Sparks, MD) produced a positive growth signal at less than 24 hours of incubation at 35°C. Gram stain of the blood bottles demonstrated large gram positive rods. The blood bottles were subcultured to 5% sheep blood agar (Becton-Dickinson). Good growth was observed after approximately 24 hours of incubation at 35°C in 5% C02. Colonies demonstrated beta hemolysis and were large, dull, slightly yellow, flat, with a granular texture, and with a spready edge. Further characterization showed the isolates to be positive for catalase and motility (agar based test).

Gas liquid chromatography (GLC)

Initial identification was attempted using GLC analysis of cellular fatty acid (CFA) content. Further analysis of the CFA content was performed using the MIDI system (MIDI Inc., Newark DE). This software compares CFA profiles of unknown organisms against a database of profiles associated with known organisms and produces a report of possible matches accompanied by a confidence score (i.e., similarity index) from zero to 1.0 where 1.0 is a perfect match. Both isolates were identified as Bacillus thuringiensis with similarity indices of .519 and .545 with no second choices.

MALDI TOF Mass spectrometry

Additional identification was performed using matrix assisted laser desorption ionization time of flight mass spectrometry analysis (MALDI-TOF MS, BioTyper, Bruker Daltonics, Billerica, MA). The isolate from lumen number 1 was identified as B. thuringiensis while the isolate from lumen number 2 was identified as B. cereus. Because both GLC and MALDI-TOF MS cannot reliably differentiate between B. cereus and B. thuringiensis the final identification of the 2 isolates was reported as a non-anthracis member of the Bacillus cereus/thuringiensis group. Supplementary Figure 1 shows MALDI TOF MS results.

Sequencing

Sanger sequencing was performed on the first 500bp of the 16S rRNA gene using procedures and reagents from Applied Biosystems Inc. (Thermo Fisher, Waltham MA). Sequence editing, text file generation, and database matching were performed using SmartGene software (Integrated Database Network Systems, Raleigh NC). Identification was determined following guidelines outlined in the Clinical and Laboratory Standards Institute (CLSI, Wayne, PA) document MM18. Species-level identification was considered acceptable when agreement between multiple reference strains and the clinical isolate was 99% or greater. Sanger sequencing on both isolates also indicated similarity to B. cereus, B. thuringiensis, and B. anthracis (ruled out due to phenotypic properties) as well as B. toyonensis, B. wiedmannii, B. proteolyticus, B. albus, B. tropicus, and B. pacificus, further highlighting the difficulty of distinguishing species within this group.

Assembly

A run of an Oxford Nanopore (ONT) MinION sequencer was performed using DNA isolated from the bacterium after growth in culture. This generated 6,117,476 reads with an N50 (weighted average) length of 2189 bp, for a total of 11.1 billion bases, with an average GC content of 36.0%. The ONT reads were assembled using the Canu assembler [1], after first downsampling the reads to 200X coverage, with 100x coverage for reads longer than 5 Kb. The Canu assembly contained 14 contigs ranging in size from 2,855 bp to 4,790,273 bp. The contigs were aligned to one another, overlapping contigs merged, and redundant ones removed, thereby creating a complete, circular assembly of the main chromosome and 2 plasmids. Separately, unpaired 151-bp Illumina reads were acquired from a metagenomics sample taken from tissue collected at autopsy and embedded in paraffin for microscopic examination. After removing human reads, 16.1 million reads remained. These reads were aligned to the Canu assembly, identifying 390,719 Illumina reads (59 Mbp) that matched the assembly. These high-quality Illumina reads were used together with the ONT reads to polish the assembly using Racon [2] and Pilon [3]. The final, polished assembly contains one complete chromosome and two plasmids, with the following lengths: 5,323,903 bp, 509,015 bp, and 295,504 bp. Re-alignment of reads to each of these molecules shows approximately equal coverage, suggesting that the ratio of plasmids to the chromosome is 1:1:1. The genome was aligned to all complete bacterial genomes at NCBI, and the closest match to the chromosome, at 99.75% identity, was B. cereus G9842 (accession NC_011772.1). The closest match to plasmid 1, at 98.3% identity, was B. thuringiensis serovar thuringiensis str. IS5056 plasmid pIS56–233 (accession NC_020394.1). The closest match to plasmid 2, at 93.9% identity, was B. anthracis str. CDC 684 plasmid pXO1 (accession NC_012591.1). The genome was then annotated with Prokka [4] and Prodigal [5], as well as with the NCBI automated pipeline PGAP. The PGAP annotation was chosen as the final annotation. This identified 5,376 protein coding genes, of which 4,611 had named functions and 765 were hypothetical proteins. Annotation also identified 518 pseudogenes, 39 rRNAs, 44 binding sites, 107 tRNAs, 43 regulatory regions, 4 ncRNAs, 2 CRISPR genes, and 1 transfer-messenger RNA (tmRNA). All assemblies and annotation have been deposited in NCBI under BioProject PRJNA591929.

To identify toxins that could have contributed to the rapid course, we compared the genes identified in our assembly with recognized B. cereus toxins [6]. Present were genes for hemolysin B subunit A (hblA), hemolysin B subunit C (hblC), nonhemolytic enterotoxin B (nheB), nonhemolytic enterotoxin C (nheC), cytotoxin K (cytK), and phospholipase C (plcA). While the pXO1 plasmid has been associated with toxicity edema factor (cya), lethal factor (lef), and protective antigen (pagA) [7], we did not find genes for those toxins.

4. Discussion

The Bacillus cereus group consists of eight species of spore-forming, gram-positive bacilli with highly similar genomes [8], which can preclude definitive identification to the species level. While species in this group are found endemic to soil, the most notable member, B. anthracis, is a zoonotic infectious agent most often spread by human contact with infected animals or animal products [9]. In contrast to B. anthracis infection, which may result in cutaneous, gastrointestinal, or inhalational disease, Bacillus species other than anthracis were previously regarded as nonpathogenic. However, these species can act as human pathogens [10], and B. cereus and B. thuringiensis are increasingly recognized as etiologic agents of non-gastrointestinal human disease, including local cutaneous and ocular infections, respiratory infections, endocarditis, [10], and sepsis [11].

Non-gastrointestinal B. cereus infections are most often seen in patients who have undergone organ transplantation [12] or with other immunocompromised states such as solid organ malignancy, end-stage renal disease, polytrauma, intravenous drug use, or hematologic malignancy [13]. Patients in this last group, particularly those undergoing induction chemotherapy, are at increased risk for rapidly-progressive infection [14–17]. Several of the reported cases with fulminant B. cereus septicemia had clinical presentations similar to our patient’s, with a rapidly progressive course, sudden onset of fever, abdominal pain, and vomiting. B. cereus sepsis was associated with hemolysis in two leukemic patients, both of whom had abdominal pain prior to rapid clinical deterioration [16]. The phenomenon may not be widely appreciated, and it has been suggested that referring to this clinical picture in neutropenic patients as ‘fulminant septicemia syndrome of Bacillus’ could increase awareness [18]. ‘Bacillus sepsis’ might be a simpler alternative.

Identifying the source of B. cereus infection can be difficult. Our patient had an indwelling line, which is associated with increased risk for disseminated infection due to colonization [19]. However, often no definitive source is identified, and many cases are attributed to the patient’s endogenous flora or gastrointestinal colonization [13, 14]. Hospital outbreaks of Bacillus sepsis have been traced to contaminated vials of calcium gluconate [19, 20], bananas (for leukemic patients) [21], tea [22], linens [23, 24], and colonization of mechanical ventilation equipment [25–27].

While the pathogenicity of Bacillus species may be attributed to any of multiple toxins and virulence factors, B. cereus produces no unique pathogenic factors allowing for distinction from other members of the group [28]. Among its potential toxins are hemolysins, phospholipases, cereulide, and enterotoxins (HBL, nhe, and cytK) [10]. Both HBL and nhe are tripartite toxins, comprised of three proteins, each encoded by a separate gene. B. cereus hemolysin BL has hemolytic, dermonecrotic, vascular permeability, and enterotoxic activity [29]. Large doses of the emetic cereulide [30] can be fatal [31]. Phylogenetic analysis has suggested a degree of relationship among B. cereus strains that cause disease [32]. Hemolysin BL shows a high degree of heterogeneity among B. cereus strains [33,34] and the presence or absence of HBL genes correlates closely with groupings of B. cereus isolates [35]. However, in studies comparing genome and clinical behavior of a number of pathologic B. cereus isolates, no relation was found between the presence of toxins and the clinical manifestations [36], highlighting the difficulty of attributing a particular clinical course to individual toxin genes. In addition to chromosomally encoded proteins and virulence factors, strains of B. cereus containing the B. anthracis plasmid pXO1 have been recovered from patients with pulmonary infections resembling inhalational anthrax [37].

Bacillus thuringiensis is a widely used biopesticide [38] and can cause food poisoning, which is often misattributed to B. cereus [39]. Early epidemiologic study in areas where B. thuringiensis was sprayed for crop protection raised concerns that immunocompromised individuals may have become infected [40], and it is now recognized as critical to monitor the strains of B. thuringiensis used in pest control to prevent human exposure to toxins [41]. Study of insecticidal properties of proteins produced by B. cereus/thuringiensis group bacteria have identified many toxins, and suggest that additional proteins not yet discovered also act as such to insects [42]. These or other proteins could contribute to toxicity during overwhelming infection in the setting of immunocompromise. Further work is needed to better identify toxins contributing to the fulminant course of B. cereus/thuringiensis sepsis in immunocompromised patients, so that appropriate therapies [43] can be developed.