A firm had multiple media fill failures. The media fill runs, simulating the filling process during production, were conducted inside an isolator. The firm used TSB (nonsterile bulk powder) from a commercial source, and prepared the sterile solution by filtering through a 0.2 micron sterilizing filter. An investigation was launched to trace the source of contamination. The investigation was not successful in isolating or recovering the contaminating organism using conventional microbiological techniques, including the use of selective (e.g., blood agar) and nonselective (e.g., TSB and tryptic soy agar) media, and examination under a microscope. The contaminant was eventually identified to be Acholeplasma laidlawii by using 16S rRNA gene sequence. The firm subsequently conducted studies to confirm the presence of Acholeplasma laidlawii in the lot of TSB used. Therefore, it was not a contaminant from the process, but from the media source.
Acholeplasma laidlawii belongs to an order of Mycoplasma. Mycoplasma contain only a cell membrane and have no cell wall. They are not susceptible to beta-lactams and do not take up Gram stain. Individual organisms are pleomorphic (assume various shapes from cocci to rods to filaments), varying in size from 0.2 to 0.3 microns or smaller. It has been shown that Acholeplasma laidlawii is capable of penetrating a 0.2 micron filter, but is retained by a 0.1 micron filter (see Sundaram, Eisenhuth, et al. 1999). Acholeplasma laidlawii is known to be associated with animal-derived material, and microbiological media is often from animal sources. Environmental monitoring of Mycoplasma requires selective media (PPLO broth or agar).
