When doing cell culture work, the term Mycoplasma has probably popped up.  

Mycoplasma is a genus of bacteria that lacks a cell wall and is notoriously difficult to treat with antibiotics. In the lab, Mycoplasma is a pervasive cell culture contaminant that you need to be constantly on the lookout for.

In the real world, Mycoplasma cause respiratory illnesses such as walking pneumonia (Mycoplasma pneumoniae), and sexually transmitted infections (Mycoplasma genitalium). It’s also responsible for an infection common to house finches, which expresses as conjunctivitis (Mycoplasma gallisepticum).

There are more than 180 species of Mycoplasma, with the most common lab contaminating species being M. fermentans, M. hominis, M. arginine, M. hyorhinis, M. orale, M. pirum, and M. salvirium.

This article gives you an easy introduction into what Mycoplasma is in the context of cell culture and its impact and prevalence within labs.

 

Article Table of Contents

The challenge of Mycoplasma detection in cell culture

Impact of Mycoplasma on cell culture and experiments

Why are Mycoplasma so pervasive in the lab?

How to deal with Mycoplasma contamination in cell culture

References

 

The challenge of Mycoplasma detection in cell culture

Mycoplasma are mollicutes. Mollicutes are a group of small parasitic bacteria that lack a rigid cell wall (Figure 1). Because they lack a peptidoglycan layer, they can change shape, though they are able to hold some structure because their membranes typically contain sterols.

Figure 1. Mycoplasma are widespread and tough to kill because they’re small and lack a cell wall.

 

Standard 0.2 µm filters prevent contamination from most bacteria which are too large to pass through these barriers. Mycoplasma, however, are really small and can squeeze through these filters leading to the spread of contamination.

Another contributing factor is that Mycoplasma are essentially invisible. Bacterial contaminants and other contaminants are visible to the naked eye. A characteristic cloudiness to the culture alerts the researcher that contamination may have occurred.

Not only do Mycoplasma not display this characteristic cloudiness, they are also invisible by microscopy. They’re too small to be visualized with standard light microscopy and they don’t get marked by most bacteriological stains, such as crystal violet, because they lack a cell wall.

The invisibility of Mycoplasma means that alternative detection methods are required. Fortunately, it is something you can test for, and we have a really helpful article that goes into more detail about how to identify Mycoplasma and some common ways to test for Mycoplasma contamination.

 

Impact of Mycoplasma on cell culture and experiments

Mycoplasma are not harmless passengers in cell culture. Rather, they alter the cells that they are growing with in several ways, from metabolism, to gene expression, to immune activation. Documented examples of Mycoplasma impacting cocultured cells include:

·         Competing for cellular nutrients and inducing cell-cycle arrest in the cells of interest (Gong et al, 1999).

·         Significantly changing the gene expression of cell lines (Miller et al, 2003).

·         Activating immune cells through binding to a protein on Mycoplasma cell surface (Shimizu et al, 2005).

·         Preventing the formation of 3D cell culture models of kidney cysts (DesRochers et al, 2015).

 

These examples demonstrate ways Mycoplasma can alter cell behavior in ways that can even go unnoticed. The result of contamination is that it can produce misleading results, reduce reproducibility and lead you to draw incorrect conclusions without realizing it right away.

Because these changes are often subtle, contamination can remain undetected while still influencing downstream experiments, which can waste time and resources in the lab.

These examples are also not an exhaustive list of the changes that Mycoplasma can cause on cell cultures, which further emphasizes just how disruptive contamination can be.

If you don’t know that your cells are contaminated, you might confuse any of these results as the impact of a biological parameter that you’re trying to study.

 

Why are Mycoplasma so pervasive in the lab?

Studies suggest that more than 10% of cell lines are contaminated with Mycoplasma (Olarerin-George & Hogenesch, 2015). Why is Mycoplasma contamination so widespread?

In part, this is because many common antibiotics don’t affect Mycoplasma. Antibiotics that target cell wall synthesis don’t impact Mycoplasma because they don’t have a rigid cell wall (Figure 1). 

Examples of common lab antibiotics that help prevent contamination but do not affect Mycoplasma are penicillins such as ampicillin and cephalosporins like cephalexin, ceftriaxone, cefuroxime, cefotaxime, and ceftazidime.

 

How to deal with Mycoplasma contamination in cell culture

Unfortunately, the most time- and resource-efficient option is usually to throw away contaminated cells and start over with a fresh, uncontaminated aliquot. But when cells are irreplaceable or too valuable to throw away, there are some things you can do.

For instance, some antibiotics will help treat cells contaminated with Mycoplasma. You also will need to address the underlying cause of contamination.

The challenge with Mycoplasma is not just that it contaminates cell culture. It’s that it often does so without announcing its presence in the first place. Your culture will usually appear healthy even when it’s not. For this reason, testing should be considered as a fundamental practice in your experimental designs, which will give you a lot more confidence in the long run.

For a more detailed guide on how to deal with cells contaminated with Mycoplasma, this article gives you more insight.

 

If you are in the position where you need to rescue a contaminated cell culture, scroll down for other research antibiotics that are effective.

 

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References

DesRochers, T. M., Kuo, I. Y., Kimmerling, E. P., Ehrlich, B. E., & Kaplan, D. L. (2015). The effects of Mycoplasma contamination upon the ability to form bioengineered 3D kidney cysts. PLOS ONE, 10(3), e0120097. https://doi.org/10.1371/journal.pone.0120097

 

Gong, H., Zölzer, F., von Recklinghausen, G., Rössler, J., Breit, S., Havers, W., Fotsis, T., & Schweigerer, L. (1999). Arginine deiminase inhibits cell proliferation by arresting cell cycle and inducing apoptosis. Biochemical and Biophysical Research Communications, 261(1), 10–14. https://doi.org/10.1006/bbrc.1999.1004

 

Miller, C. J., Kassem, H. S., Pepper, S. D., Hey, Y., Ward, T. H., & Margison, G. P. (2003). Mycoplasma infection significantly alters microarray gene expression profiles. BioTechniques, 35(4), 812–814. https://doi.org/10.2144/03354mt02

 

Nikfarjam, L., & Farzaneh, P. (2012). Prevention and detection of Mycoplasma contamination in cell culture. Cell Journal, 13(4), 203–212.

 

Olarerin-George, A. O., & Hogenesch, J. B. (2015). Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI's RNA-seq archive. Nucleic Acids Research, 43(5), 2535–2542. https://doi.org/10.1093/nar/gkv136

 

Shimizu, T., Kida, Y., & Kuwano, K. (2005). A dipalmitoylated lipoprotein from Mycoplasma pneumoniae activates NF-κB through TLR1, TLR2, and TLR6. The Journal of Immunology, 175(7), 4641–4646. https://doi.org/10.4049/jimmunol.175.7.4641

 

Uphoff, C. C., Denkmann, S. A., & Drexler, H. G. (2012). Treatment of Mycoplasma contamination in cell cultures with Plasmocin. Journal of Biomedicine and Biotechnology, 2012, 267678. https://doi.org/10.1155/2012/267678

 

 

 

 

 

 

 

 

 

 

 

 

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antibiotic resistance antibiotics cell culture mycoplasma Simon Currie

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