STRUCTURAL ORGANISATION OF THE SURFACE OF BACILLUS CEREUS SPORES
The interest in the spore-forming bacteria Bacillus cereus is due to their widespread occurrence and their ability to cause various diseases, mainly of the gastrointestinal tract. Spores of pathogenic microorganisms, including B. cereus, pose a great challenge to medicine, pharmacology and food industry because of their resistance to various environmental factors. The properties of the spores are determined by their ultrastructure. The basic structure of spores is conservative and consists of the exosporium, shell, outer membrane, cortex, inner membrane and core . However, the outer layers of the spores, including the exosporium, vary between species and strains, allowing the features of their components to be compared by electron microscopy.
Exosporium is a structure characteristic of the spores of the B. cereus, B. thuringiensis, B. anthracis and consists of an outer villous layer, an inner paracrystalline layer, a hexagonally perforated basal layer and contains proteins, lipids and polysaccharides [2–4]. The exosporium has long outgrowths that promote spore adhesion . The exosporium is located at a considerable distance from the shell (e.g. 500 nm in B. anthracis . The electron-transparent region between the basal layer of the exosporium and the spore shell (the intermediate exosporial space) in some strains contains structures that form additional surface layers. The exosporium plays an important role in resistance, adhesion, proliferation and spore germination. The exosporium is a filter that allows the germination inducers – alanine or inosine – but not proteolytic enzymes or antibodies, to pass through . The spores of the mutant B. cereus which has increased lysozyme resistance and altered germination ability, forms a double exosporium . Important for structural studies of the exosporium is its naturally crystalline nature. Exosporium can be used as a model for creating self-organizing supramolecular, nanoscale structures. It has been shown that the main protein of the ExsY basal layer can assemble into ordered two-dimensional structures that mimic the exosporium. Self-assembly, probably, plays an important role in exosporium construction . Using electron microscopy and computer image analysis, we have previously obtained data on the fine structure of the exosporium of the reference strain B. cereus .
It was of interest to study exosporium in spores of strains of different origin and natural and clinical isolates by means of electron microscopy and computer image analysis. These strains have not been studied until now. Particular attention is paid to the external structures of B. cereus spores (exosporium and exosporium space) of these strains and their organization as the elements providing for surface properties. The aim of the present study is to determine the lattice parameters of the basal exosporium membrane and to compare the surface structures of spores of strains from different ecological niches.
MATERIALS AND METHODS
Strains and nutrient media
A collection reference strain NCTC 8035 and natural strains (115\079, 131\079) of B. cereus were used. The strains were grown on LB agarized nutrient broth at 28 °C for 96 hours. Clinical isolates were obtained from patients with ulcerative colitis (UC) treated in A.N.Ryzhikh State Scientific Centre for Coloproctology, (Moscow, Russia) of the Ministry of Health of the Russian Federation, designated as B. cereus SCCC 1208, B. cereus SCCC 19/16. The following strains were also investigated: strain 169 of B. cereus isolated from abdominal secretions of a patient with UC; strain 177 of B. cereus isolated from lumen feces; strain 172 of B. cereus isolated from lumen feces; strain 214/18 of B. cereus isolated from the operative wound of a patient with UC; strain 239/18 of B. cereus isolated from the blood of a patient with confirmed sepsis; strain 223/18 of B. cereus isolated from the blood of a patient with UC; strain isolated from the operative wound of a UC patient; strain 239/18 B. cereus isolated from the blood of a UC patient with a confirmed diagnosis of sepsis; strain 223/18 B. cereus isolated from the abdominal cavity of a UC patient strain 181 B. cereus isolated from the operative wound of a UC patient. The growth medium was blood agar.
Transmission electron microscopy
After 96 hours of cultivation the bacteria were washed off the nutrient-dense medium and washed out with distilled water. To study spores by negative contrast, the spore suspension was applied to copper grids covered with a formvar film and stained with 1% aqueous uranyl acetate solution or 2% aqueous ammonium molybdate solution. To obtain ultrathin sections, the samples were fixed using the Ito-Karnowski method . The material was then fixed in 1% OsO4 solution on 0.2 M cacodylate buffer and in 1% uranyl acetate solution on 0.2 M maleate buffer. The material was dehydrated in alcohols with concentrations of 50°, 70°, 96°, and 100°. The material was then placed in a 100° alcohol mixture with LR White resin and then in pure LR White resin. The material was transferred to gelatin capsules which were placed in a thermostat at 56 °C. Sections were obtained on an LKB III ultratome (LKB, Sweden) and contrasted with a 1% solution of uranyl acetate in 70° alcohol and citric acid lead. Negatively stained spores and sections were studied in a JEM 2100 electron microscope (Jeol, Japan) at an accelerating voltage of 160 kV. Image analysis was performed using FemtoScan Online software (Center for Advanced Technologies, Moscow, www.nanoscopy.ru) .
Electron microscopic examination of the spores of the reference strain, natural strains and clinical isolates of B. cereus was carried out using negative contrast and ultrathin sections. Figures 1–7 show mature spores of the reference strain 8035, natural strains and clinical isolates of B. cereus. The reference strain 8035 has an ultrastructure characteristic of B. cereus (Fig.1).
A cross section shows the following structures: the exosporium as an electron-dense outer layer, the electron-transparent exosporial space separating the exosporium from the electron-dense spore shell, the electron-transparent cortex, and the core at the centre of the spore. These results provide an overview of the spore structure of B. cereus. All the strains studied (natural and clinical isolates) had a similar basic ultrastructure, but also had their own specific features. In the spore of the natural strain B. cereus 131, a cross-section of the exosporium shows multiple membrane inclusions repeating the course of the exosporium (Fig.2).
In the clinical isolate B.сerеus 19, a longitudinal section shows that the spore is located at one pole of the exosporium, a loosely adhering sheath, and most of its internal electron-transparent space is filled with chaotically arranged fragments of membrane structures (Fig.3).
Transverse sections of the spores of clinical isolates show that the exosporium contains structures not present in the spores of the reference strain. In Fig.4 a, b, c, an additional layer of the second exosporium can be seen in clinical isolates 214, 177 and 239.
Thus, the surface spore layers of natural strains and clinical isolates were found to be thicker and, possibly, less permeable than those of the reference strain. Such feature may protect the spore from various bactericidal factors. In the case of natural strains, these may be weather-related influences, whereas clinical isolates must resist the antibacterial defense mechanisms of the host. The reference strain 8035, grown under laboratory conditions, is not exposed to such influences and has a more simplified spore surface.
Electron microscopic examination of the spores of clinical and natural isolates was also carried out using the negative contrast method. Figures 5, 6 show spores and their fragments negatively contrasted with 2% ammonium molybdate. Previously, spores of the reference strain 8035 were examined by negative staining .
The exosporium of the examined spore strains, same as in strain 8035, was revealed as an electron-transparent bilayer sacculus surrounding an electron-dense spore. The peripheral region of the exosporium was examined to reveal the ultrastructure. Figure 5 shows areas of the exosporium in natural strains B.сerеus 114 and 115 with clearly distinguishable villous and basal layers, and a similar area of the exosporium in clinical isolate 239. Long spore outgrowths are visible on the spore surface of strains 114 and 115.
At high magnification, all images show the hexagonal packing of the subunits of the basal membrane. However, the structure of this membrane is not clearly visible.
To obtain a clear picture of the exosporium spores of the reference strain 8035, Fourier filtering was previously applied and a noise-free image showing hexagonal packing and holes was obtained. Similar work was carried out with exosporium strain 239 (Fig.6). Measurement of the reflex period showed no significant changes in the pore spacing in the hexagonal packing of strain 239 exosporium compared to the reference strain: strain 8035 had a length of 5.6 nm, 6.2 nm and 4.7 nm. In Fig.6c, the length is 5.2 nm, 5.5 nm, 5.8 nm. The average value is the same for the reference strain and strain 239, 5.5 nm.
Figure 7 shows a single-layer fragment of the exosporium of the clinical isolate B. сerеus 214 showing similarities with the exosporium of the other clinical isolate B.сerеus 239.
The study revealed common and different features between the reference strain and the natural and clinical isolates. In particular, differences in the surface layers of the spores of natural strains and clinical isolates were found – as compared to the reference strain, they are thicker and apparently better protect the spores from external penetration. An additional second exosporium layer was detected in the clinical isolates. Using Fourier filtration, common features in the hexagonal packing of the exosporium were identified – the distance between the pores of one layer is, on average, 5.5 nm.
The obtained results can be used to understand the characteristics of B. cereus spores from different ecological niches and their sensitivity to sporocidal factors.
The study was completed with the financial support of the RFBR and the London Royal Society No. 21-58-10005, and RFBR, Project No. 20-32-90036. This research was carried out with financial support from the Foundation for the Promotion of Innovation, Project No. 71108, and Agreement No. 0071108.
PEER REVIEW INFO
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Declaration of Competing Interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.