Bacterial capsule, colony morphology, functions, and its relation to virulence and diagnosis

Microorganisms possess many virulence factors that are usually decided by their genetic makeup. Not many virulence determinants of bacteria are phenotypically expressed. Capsule is one such bacterial organelle, which displays many functions that include adherence, resistance to immune clearance, protection against environmental factors, and many others including the typing of bacteria based on their specific capsular antigen and rapid diagnosis of capsulated bacterial infections using monoclonal/polyclonal anticapsular antibodies.

Keywords: Bacterial capsule, Functions of slime and capsule, virulence determinants

Identifying the bacteria isolated from clinical specimens and performing their antimicrobial susceptibility testing form a significant part of the duties performed in a clinical microbiology laboratory. The factors that help a microbiologist to identify the bacterium include the nature of isolation (pure and mixed culture), specimen source, growth requirements for isolation (simple, enriched, or selective media), colony characters, microscopic characters, staining reactions, and biochemical features. Among these, bacterial colony morphology plays a key role in the preliminary identification of the bacteria. A bacterial colony is formed by a single bacterial cell that divides by binary fission to form thousands of clones. This is the reason we call each bacterium a single colony forming unit (CFU).

Culture medium plays a key role in the demonstration of bacterial colony characters and the real morphology of the bacteria. The media consisting of simple ingredients and without any inhibitory substances are suitable for describing the colony characters of a bacterium (nutrient agar). Bacterial colonies vary in shape, size (measured in diameter), odor, color (pigmented), texture, and degree of adherence to the media (pitting and crusting). Different bacteria show different colony morphologies that include those such as rhomboidal in shape (e.g., Pseudomonas spp.), large mucoid colonies (e.g., Klebsiella spp.), colonies with wavy edges (e.g., Bacillus anthracis), swarming colonies (e.g., Proteus spp.); the colonies can be mucoid (M colonies), smooth (S colonies), and dry (R colonies). ^[1] An M colony appears water-like, glistening, and confluent (no individual or separated colony) and is a character of a bacterial colony that produces slime or a capsule [Figure 1]. S colonies are recognized by their moist nature, and are indicators of freshly isolated wild bacterial strains. R colonies are rough, dry, granulated, and mutant types of bacteria that lack most of the surface proteins including the capsule and lipopolysaccharides. R colonies are formed by bacteria that are usually avirulent. The ability to show variations in both smooth-rough (S-R) ways and from rough to smooth (R-S) colonies has also been observed in bacteria. The rough colonies formed on blood agar, which on Gram stain reveal gram-positive bacilli that form spores (aerobic spore-bearing bacilli), are usually ignored as laboratory contaminants. Large encapsulated bacteria appear viscous and shiny, whereas nonencapsulated bacteria form small and dull colonies. ^[2]

Glycocalyx is the term that was used to refer to all polysaccharide-containing substances outside the cell wall. The capsules are among the first known bacterial virulence determinants, which were initially demonstrated by Griffith in 1928 in his rat experiments. This experiment has proved that suspension of smooth colonies of bacteria are able to produce capsule that can kill mice, whereas suspension of the rough colonies of bacteria devoid of any capsule caused no mortality. Slime is an extracellular polymer loosely attached to the cell wall that can be easily washed off, whereas capsule is a compact, thick, and gelatinous structure adhered to the cell that is made of glycocalyx polysaccharides, glycopeptides, etc. Capsules are usually polysaccharide in nature, may be homo-/monopolymers of glucans, dextrans, and levans or heteropolymers of hexose and pentose sugars plus ribitol/glycerol/other sugar alcohols; and capsule can also be composed of phosphodiesterases. Capsule is also made of repeating the oligonucleotide of more than one monomer. Many exopolysaccharides are acidic in nature due to the heavy carbonyl groups, either from acidic sugars such as uronic acids/neuraminic acids or from nonsugar subunits such as pyruvyl, acetyl, and formyl groups. The capsules are either synthesized at the level of cell membrane or extracellularly, as done by Streptococcus mutans using extracellular enzyme glucosyltransferase in the oral cavity. Individuals infected with capsulated bacteria like Streptococcus pneumoniae, Klebsiella pneumoniae, Neisseria meningitides, Haemophilus influenza, Bacillus anthracis, Yersinia pestis, and others show anticapsular antibodies in their serum. The capsule is impermeable to biological dyes routinely used; therefore, they cannot be stained. Negative staining, which colors the background, is employed to demonstrate the capsule. India ink and nigrosin are the two stains used for staining the background and later, a simple stain like safranin or crystal violet is applied to stain the bacteria before observing them through the microscope. ^[3] The capsule is viewed as a clear halo surrounding the bacteria [Figure 2]. The bacteria showing the capsular antigen can be typed using specific monoclonal/polyclonal anticapsular antibodies. Quellung test is a serological test done for the demonstration of capsule wherein the bacteria are probed with specific anticapsular antibodies and are later stained and observed under the microscope. The capsule is seen as an increase in the halo surrounding the bacteria. This test is also known as capsule swelling reaction. Capsular material/antigen of many microorganisms is synthesized in abundance, and is shed into the surrounding environment in vitro and in vivo. This antigen can be detected in various body fluids (serum and spinal fluid) by various laboratory diagnostic methods like electrophoresis and agglutination methods, which can aid in rapid diagnosis. ^[4]

Slime protects the cell against drying, helps trap nutrients near the cell wall, and binds the cells together. Slime may also help the bacteria to adhere to objects both in vivo and in vitro. The capsule prevents the bacteria from getting phagocytized, thereby acting as antiphagocytic in nature. The capsule consists of high molecular weight polysaccharides that make the bacteria very slippery and difficult for white blood cells to phagocytize. The capsule helps the bacteria to adhere to surfaces, camouflages the bacteria from the immune system by mimicking the host tissues, and makes the bacteria resistant to complement invasiveness. A capsule protects cells from desiccation and toxic metabolites in the environment (heavy metal ions and free radicals). It also helps in promoting the concentration of nutrients at the bacterial cell surface because of its polyanionic nature. To establish itself and to cause infection, the bacteria take the help of the capsule to adhere to the cell and mucus membranes. The capsule also empowers the bacteria in becoming resistant to bactericidal action of complement and serum antibodies. It also acts as a diffusion barrier against antibiotics and contributes toward resistance mechanisms. ^{[5],[6],[7],[8],[9].}

Capsules are the first known virulence determinants of microorganisms. Of the many virulence factors, capsule is the one that is phenotypically expressed. They are genetically coded and are also transferable from one species to the other. Being antigenic in nature and present both as an attachment to cells and extracellularly, the capsule stimulates the immune system to produce anticapsilar antibodies that can be utilized for the rapid diagnosis of infections.