Identification of a New Pathogenicity Island Within the Large pAH187_270 Plasmid Involved in Bacillus cereus Virulence

Abstract

Objectives: Bacillus cereus is responsible for food poisoning and rare but severe clinical infections. The pathogenicity of B. cereus strains varies from harmless to lethal strains. The objective of this study was to characterize three B. cereus isolates isolated from the same patient and identify their virulence potentials.

Methods: Three isolates of B. cereus were isolated from various blood samples from a patient who developed sepsis following a central venous catheter infection. The three isolates were compared by WGS, genotyping and SNP analysis. Furthermore, the isolates were compared by phenotypical analysis including bacterial growth, morphology, germination efficacy, toxin production, antibiotic susceptibility and virulence in an insect model of infection.

Results: According to WGS and genotyping, the 3 isolates were shown to be identical strains. However, the last recovered strain had lost the mega pAH187_270 plasmid. This last strain showed different phenotypes compared to the first isolated strain, such as germination delay, different antibiotic susceptibility and a decreased virulence capacity towards insects. A 50- kbp region of pAH187_270 plasmid was involved in the virulence potential and could thus be defined as a new pathogenicity island of B. cereus.

Conclusions: These new findings help in the understanding of B. cereus pathogenic potential and complexity and provide further hints into the role of large plasmids in the virulence of B. cereus strains. This may provide tools for a better assessment of the risks associated with B. cereus hospital contamination to improve hygiene procedure and patient health.

Keywords: Bacillus cereus; clinical infection; mega-plasmid; pathogenicity island; virulence.

Figure 1

(A) M13-PCR fingerprint patterns, Lane 1: 1 kb DNA ladder. Lane 2: reference strain B cereus ATCC14579. Lane 3 to 5: S94, S20, S95 B cereus strains. (B) Visualization of core and accessory genomes of patient isolates S94, S20, S95, along with reference strain B cereus AH187 and mega-plasmid pAH187_270, using RAxML-generated phylogenetic tree and presence/absence table generated by Roary. Red box highlights absence of pAH187_270 genes in strain S95. (C) Proksee visualization of strains S94, S20, S95 aligned to the plasmid pAH187_270 nucleotide sequences. Key biomarkers are identified within the plasmid (green arrows), as well as the ces encoding gene. (D) Overview of the PAI. The plasmid region from 125,000 to 192,000bp of pAH187_270 contains all of the plasmid-based biomarkers (blue arrows) and several transposases/recombinases (green and red arrows, respectively). The average GC content is indicated above the genes within the PAI (light green) and in the surrounding regions of the plasmid (purple).

Figure 2

(A) left: Determination of growth curves of B cereus strains (S94, S95, S94Δpai) by measuring the OD_600nm in BHI medium at 37°C during 6.5 hours. Each point is the mean of four independent experiments. Vertical bars indicate standard errors. Right: representative images of microscopic observation of the strains at DO_{600 nm} = 0.3. (B) B. cereus strains) were plated on BHI plates to obtained single colonies. Left: the sizes of 25 colonies per plate were measured. The results indicate the mean of three independent experiments with standard errors; Right: representative images of the strains. (C) Germination rate efficiency measured over 55 minutes. The spores of the strains were incubated in BHI and at each time point, the remaining heat resistant bacteria (spores) were measured and normalized to the initial spore values at t0. The decrease in spore value indicate the rate of germination. The results indicate the mean of three independent experiments with standard errors. (D) Mortality rate of G mellonella injected with increasing doses of the strains was assessed 24 hours post-infection. Each dot represents the data for 10 larvae. Several dots are overlapping for the S95 strain.