FIG. 1. Developing Mycobacterium smegmatis biofilm (5 days old) in 12-well cell culture cluster plates using modified M63 liquid media.
FIG. 2. Mature Mycobacterium smegmatis biofilm (7 days old) in 12-well cell culture cluster plates using modified M63 liquid media.
Microorganisms have many ways to ensure their survival, such as sporulation and biofilm formation. In nature, most microbes live as communities in biofilms, a conglomeration of bacteria and other microbes embedded in a self-produced and secreted matrix of extracellular polymeric substances (EPS) (1). The EPS can be composed of polysaccharides, proteins, nucleic acids, and lipids. The biofilm functions as a protective hydrated barrier between the bacterial cells and their environment. It facilitates survival under harsh conditions and environmental insults such as ultraviolet radiation, physicochemical stresses, desiccation, and insufficient supply of nutritive resources (3).
Mycobacterium smegmatis are aerobic nonmotile bacteria, which are characteristically acid fast and grow well as biofilms (6). As shown in Fig. 1 and 2, the biofilms produced by M. smegmatis are surface pellicles (i.e., they appear at the air-liquid interface). Unlike other bacterial biofilms which synthesize EPS composed predominantly of polysaccharides, mycobacterial EPS contains large amounts of mycolic acids (waxes), which are also found in the cell envelope of mycobacteria. These mycolic acids are hydrophobic and result in the organism growing as a surface pellicle on the aqueous medium (5).
The maturing biofilm surface pellicle at day 5 after inoculation has a characteristic ridge-like appearance (Fig. 1). At day 7, the complex network of ridges appears to be more pronounced (Fig. 2). However, since Mycobacterium is a slower growing organism, the differences between the 5-day and the 7-day biofilms are difficult to ascertain. The air-liquid interface, where these biofilms form, can be seen in each figure in the adjacent well. The biofilm is pulling away from the sides of the well revealing the bulk fluid sequestered underneath the surface pellicle. The fluid has a deeper yellow coloration after 7 days (Fig. 2) compared to after 5 days (Fig. 1) due to the accumulation of biowastes.
Prior to inoculation, M. smegmatiscells were grown on 7H11 agar plates supplemented with 10% oleic acid, albumin, dextrose, and catalase. Purity was confirmed by acid-fast staining (5). Bacterial colonies were inoculated in a modified M63 liquid media, a synthetic media consisting of vital chemicals, metal ions, and other nutrition such as casamino acids and glucose, prepared under sterile conditions, to match the 0.5 MacFarland turbidity standard. Four milliliter aliquots of the liquid suspension were distributed into wells of a 12-well cell culture cluster plate and incubated without agitation at 37°C for 7 days. Digital photographs were taken using a 10.0 megapixel digital camera.
Several species of Mycobacteria can cause diseases in animals and humans. M. tuberculosis is the causative agent of tuberculosis, while leprosy is due to the pathogen M. leprae. M. smegmatis and M. avium are both opportunistic pathogens (8). Although M. smegmatis has been shown to easily form thick biofilms (6), other species of Mycobacteria are also able to form biofilms under specific conditions. Mycobacterium avium occurs in the environment and can cause disease in humans and animals as an opportunistic pathogen. It has been demonstrated that clinical isolates of M. avium from AIDS patients can form biofilms on Polyvinyl Chloride (PVC) plates (2). Recently, M. tuberculosis has also been shown to form biofilms in petri dishes (7).
Since bacteria in biofilms are more resistant to antibiotics (3), biofilm formation on in-dwelling medical devices and damaged tissue, such as catheters and prosthetic joints and heart valves, is an ongoing medical concern (4). Recent studies support the possibility that pulmonary tubercles formed by Mycobacterium tuberculosis may be constructed as biofilms and may be part of the reason why long-term therapy is required to eliminate a tuberculosis infection (7).
The air-liquid phase biofilms formed by Mycobacterium smegmatis are distinct and easy to visualize. They are a great example of bacterial biofilms to use in the classroom.
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