What is Mycoplasma?
11th January 2019
By: Daniel Cozens - Biosafety Scientist,
Mycoplasmas are the smallest self-replicating organisms currently known to science. Common in nature, they are a group of simple bacteria characterized by a lack of a cell wall and a small genome. Many Mycoplasma species are found in the respiratory tracts of animals, where they have been associated with disease. For example, Mycoplasma pneumoniae is a leading cause of respiratory infections in humans (1).
As well as clinical infections, mycoplasmas are frequent contaminants of mammalian cell culture for a myriad of reasons.
- They are capable of evading host cellular defences in order to survive within cells.
- Their small size means they cannot be observed by light microscopy and are capable of passing through anti-bacterial filters.
- Many common antibiotics used to protect against bacterial contaminants during cell culture are ineffective due to a lack of cell wall in the bacterium.
- Neither the presence of the contaminating bacteria or the consequential morphological changes to the cell can be readily identified visually using light microscopes, making contamination difficult to detect.
This has resulted in Mycoplasma species being a widespread source of cell culture contamination, and mycoplasmas are a major issue for the biopharmaceutical industry.
Effects of Mycoplasma Contamination
Due to their inability to produce several key molecules needed for life, Mycoplasma species must live as parasites within host cells in order to replicate. The bacteria are capable of invading mammalian cells to gain access to biosynthetic precursors and other nutrients. This process can drastically alter the metabolism of the host cell. Furthermore, contaminated cells can show induced chromosomal alterations and variation in gene expression. These changes can drastically reduce yield and compromize the safety of biopharmaceutical products.
Testing for Mycoplasma
Safety regulations require the demonstration of an absence of detectable levels of mycoplasmas in the product through mandatory testing. Outsourcing this testing can guarantee that it is performed to the specification set by regulatory standards. Many of the guidelines outline the use of both the agar and broth method and in vitro indicator cell culture testing as the current gold standard. However several authorities, including the European Medicines Agency (EMA) (2) and US Food and Drugs Administration (FDA) (3), also allow for nucleic acid-based technologies (NAT-based) methods, such as PCR, following appropriate validation of the assay.
Sartorius Stedim BioOutsource (SSBO) offer a variety of tests for the detection of mycoplasmas and can provide guidance to clients on which test is most suitable for their test material.
Qualification of validated mycoplasma detection assays must be performed in the presence of test material prior to testing. This is to detect any inhibition of mycoplasma growth caused by the test material (mycoplasmastasis) and this involves spiking the test material with low levels of mycoplasmas, such as M. pneumoniae or Mycoplasma orale, and demonstrating recovery.
Agar and Broth Method
The direct culture method allows the detection of a large range of Mycoplasma species. The test material is used to directly inoculate agar and broth specifically designed to support growth of mycoplasmas. Agar plates are incubated for 14 days. Broth cultures are grown for 21 days, with subcultures taken to inoculate further agar plates. These plates are then observed microscopically for the presence of Mycoplasma colonies. Positive controls of two mycoplasma species are also inoculated during the test. The exact species used is dependent on the type of sample being tested.
In Vitro Indicator Cell Culture
For the indirect indicator cell culture method, Vero cells or another appropriate cell line, are inoculated with the test material and incubated for three days. Cells are subsequently fixed and stained with fluorescence dye that can bind to DNA, such as Hoechst stain or 4’6-diamidino-2-phenylindole (DAPI). Cells can then be observed under fluorescence microscopy. Mycoplasma contaminants are identified by their characteristic extra-nuclear cluster or filamentous staining patterns within cells. Through this assay, non-cultivable species which are reliant on mammalian cells for growth can be detected.
The combination of the direct and indirect approaches allows for the identification of a wide range of Mycoplasma species. The benefit of these combined methods is that due to the presence of an incubation period, mycoplasmas can replicate, increasing the sensitivity of the assay. However this incubation period also lengthens the testing period, delaying the time by which any contamination can be confirmed.
Rapid Mycoplasma PCR
A number of authorities, including the EMA and FDA, accept alternative NAT-based testing as a replacement to the traditional direct and indirect methods of mycoplasma detection. The benefit of these methods is the rapid testing time, allowing for faster decision making and quicker quarantine of contaminated material during biopharmaceutical manufacture. For the assays to be validated they must first be shown to be comparable or better than traditional testing and provide broad detection of mycoplasma species.
SSBO provides a fully validated and qualified real-time PCR assay for the detection of mycoplasmas which is compliant with European Pharmacopoeia section 2.6.7. For this assay, nucleic acid is extracted from any mycoplasma cells present within the sample, both free organisms and within host cells. Following extraction and purification, the DNA undergoes real-time PCR to identify and quantify low levels of target sequences specific for Mycoplasma. Test material spiked with Mycoplasma Discriminatory Positive Control is also run in the SSBO assay. This is used to identify any inhibition caused by the sample in the detection of mycoplasma.
Testing for mycoplasmas is essential for both ensuring the safety and efficiency of biopharmaceutical manufacture and the detection and quarantine of contaminated material to prevent the spread of the organism within the production facility. We can provide both testing using the direct and indirect method and a fully validated real-time PCR for rapid testing of test material. Testing follows the specifications set by several regulatory bodies including the EMA and FDA, providing the documentation needed for the release of biopharmaceuticals onto the market.