The structure of the appendages on spores of bacillus cereus
Bacillus cereus is a ubiquitous bacillus species. B. cerеus is known to cause various diseases, mainly associated with damage to the gastrointestinal tract (GIT) with symptoms of diarrhea and vomiting. B. cerеus can be an etiological agent of meningitis, pericarditis, pneumonia, eye diseases which accompanies wound infections. New data on the structure of outgrowths of B. cerеus bacteria are presented on the basis of the experimental data obtained with the aid of the transmission electron microscopy and computer image analysis.
An important characteristic of the bacilli is the ability to sporulate. Spores are one of the most resistant forms of bacteria. The importance of spores is due to their thermal stability, UV resistance and resistance to various environmental factors. From this point of view, spores pose a danger to the food industry and medicine. Currently, the morphology of most spore-forming bacteria has been studied. Along with the basic traits characteristic of a certain species, a number of strains exhibit properties that depend on the source of isolation. Strains of B. cerеus isolated from the environment, food products and obtained from patients may be different in their characteristics. The morphological features of bacilli appear at the stage of sporulation. Spores of different species and strains have distinctive features that are absent in vegetative cells.
DesRosier and Lara  showed that B. cereus have the appendages, similar to pili, of 6.8 nm diameter and variable lengths. They are proteinaceous and highly stable. Application of the labeled antibodies proved a presence of common antigens in pili and exosporium. Spores appendages of various morphological types were found in B. thuringiensis by Smirnova et al. .
Recently, the spore protein filamentous appendages of two morphological types were found in B. cereus . The authors found endospore appendages (Enas) in the NVH 0075-95 B. cereus strain, which causes food poisoning. Enas has been shown to represent a new class of spore-specific pili of gram-positive bacteria. Enas are stabilized by disulfide bonds, making the flexible pili heat-resistant and insensitive to dryness and chemical damage.
The appendages of spores are of interest because of the adhesive ability revealed in them. Due to presence of appendages, spores can attach to biological and inert surfaces that are important for medicine and the food industry. The adhesion capacity of spores and their piling vary from strain to strain and is the greatest in spores with long projections .
It is interesting to study the features of appendages in B. cereus strains of various origins to assess the ability of spores to attach to the equipment used in various industries and medicine. The data on the structure and properties of the appendages of spores appendages are important for choosing a method for combating spore-forming bacteria. Nowadays, the features of the spiral-like packing of subunits of spore appendages of B. cereus – the nanostructures specific for spores – are being investigated.
MATERIALS AND RESEARCH METHODS
Research materials were B. cereus strains, numbers and sources of isolation as well as the media on which the inoculation was made. We used a reference NCTC 8035 strain, natural (137 \ 0719, 114 / 0719, 115 \ 079, 131 \ 079) and clinical isolates of B. cereus. The strains were grown in nutrient broth LB at 28 °C for 12–96 hours. Clinical isolates were obtained from the luminal feces of patients with ulcerative colitis (UC) at the A.N.Ryzhikh State Scientific Center of Coloproctology Ministry of Health of the Russian Federation, designated as SCCC 1208 B. cerеus and SCCC 19/16 B. cerеus. The following strains were also investigated: strain 169 B. cereus isolated from the discharge of the abdominal cavity of a patient with UC; strain 177 B. cereus isolated from luminal feces; strain 172 B. cereus isolated from luminal feces; strain 214 / 18 B. cereus, isolated from the surgical wound of a patient with UC; strain 239 / 18 B. cereus, isolated from the blood of a patient with UC and a confirmed diagnosis of sepsis; strain 223 / 18 B. cereus isolated from the abdominal cavity of a patient with UC; strain 181 B. cereus isolated from a surgical wound of a patient with UC. The growing medium was blood agar. The strains were subcultured onto a solid nutrient medium NBY and grown at 30 °C.
TRANSMISSION ELECTRON MICROSCOPY
After 96 hours of cultivation, the bacteria were washed off from a dense nutrient medium, rinsed with distilled water and fixed with formalin. To study spores by negative contrast method, a spore suspension was applied onto copper grids covered with a formvar film, stained with 1% aqueous solution of uranyl acetate and 2% aqueous solution of ammonium molybdate.
Negatively stained spores were studied using a JEM 2100 electron microscope (Jeol, Japan) at an accelerating voltage of 100 kV.
The quantitative analysis of the obtained data was processed using the FemtoScan Online software [7, 8]. To determine the degree of flexibility of the appendages, we carried out a measurement of the persistent length, which is widely used in the study of the mechanics of macromolecules . The estimated value of the persistent length Lp was also determined by the approximate formula Lp ~ R2/L, where L is the contour length of the polymer chain (appendage), R is the distance between the ends of the polymer chain (appendage).
An electron microscopic study of the B. cereus strain spores was carried out using the negative contrast method.
As can be seen from the presented images of transmission electron microscopy with negative contrast, an exosporium is observed in the form of an electron-transparent bag (Fig.1) surrounding the spore, and spore appendages (Fig.2). The number and length of the spore appendages depend on their belonging to the strain (Fig.3). The length of the appendages varies, reaching 11 microns and the diameter is about 9–10 nm. At high magnification it can be seen that they differ in morphology. Large tubular appendages were found to have a canal inside. In the transmission electron microscopy images it is noticeable that the channel is filled with a contrast agent. At high magnification, a spiral pattern is revealed (Fig.3a). In some pictures, the appendages are in contact with each other. In addition to tubular, thinner flexible appendages are observed. The appendages originate from the exosporium, but, possibly, from the shell too. They are distributed individually or in bundles.
A quantitative analysis of the obtained images of spores showed that the appendages about 1 μm long have, as a rule, the form of rectilinear rods. Slight curvature is noticeable in case of long appendages 1–5 µm long. The obtained value of the persistent length is 6 ± 0.9 μm. The calculated persistent length indicates that the appendages are a rigid polymer chain. It should be noted that, in spite of the applied procedure of sample preparation which includes deposition on a solid substrate, drying, and evacuation, practically all of the appendages have an integral character without visible damage. In the view field near bacteria, fragments of appendages isolated from bacteria are not detected or their number is insignificant, which indicates a high stability of the appendages. The destruction of the appendages during preparation of samples and subsequent observation in an electron microscope practically did not occur. The appendages originate directly from the edge of the bacterial spore to a distance of 1–10 µm. These facts also indicate a high mechanical strength of the appendages. The observed diameter of the appendage channel is about 1.5 nm.
The pitch of the spiral packing is about 4 nm and the observed angle of rotation of the helix is about 77 degrees, which corresponds to the 13 degrees angle of spiral rotation in relation to the cross-sectional plane. With a helix pitch of 4 nm and an appendage diameter of about 9 nm, the calculated helix angle is also about 13 degrees (Fig.4c).
According to the obtained data, the previously unexplored B. cereus strains have spores appendages typical of these bacilli. These strains differ in the number of spore appendages. The smallest number of appendages is characteristic of the natural strains. Clinical isolates have more spore appendages. The piling of the spores of clinical isolates is expressed in different ways in different strains. Tubular appendages predominate. We have previously shown that in entomopathogenic bacilli related to B. cereus the appendages of this type are responsible for adhesion to erythrocytes and cause a hemagglutination reaction . It is likely that in B. cereus these structures perform a similar function.
The results obtained in this work indicate the strength of the appendages, which is characteristic of spores structures. The resistance of the appendages to external influences obviously complicates the procedure to clean the environmental objects from spores. Along with the practical significance, the study of appendages is important for understanding the features of assembly and organization of gram-positive bacteria pili, as a new class of nanostructures. ■
This work of I.V.Yaminsky was supported by the Russian Science Foundation, project No. 20-12-00389, and A.I.Akhmetova within the projects No. 20-32-90036 and No. 21-58-10005 of the Russian Foundation for Basic Research.