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. 2023 Jan 4;75(2):135–141. doi: 10.1007/s10616-022-00567-7

Identification of cell culture contamination by an unusual species of Mycoplasma related to the M. mycoides cluster

Jose Antonio Carrillo-Ávila 1,✉, R Aguilar-Quesada 1, G Ligero 1, S Panadero-Fajardo 1, M V Santos-Pírez 1, P Catalina 1
PMCID: PMC10030751  PMID: 36969572

Abstract

Mycoplasma contamination is a significant problem in cell culture replication and maintenance. From more than 200 known species, a limited number of Mycoplasma species have been detected in cell cultures, representing new species or variants that can escape detection systems. A qPCR commercial kit was used for Mycoplasma detection in cell cultures. Furthermore, an amplified Mycoplasma species was sequenced and summited for sequence assembly, clustering, and evolutionary analysis study. Our work has identified a new and unusual variant or species of Mycoplasma that possesses a high degree of homology with species related with M. mycoides cluster. This variant is usually associated with cattle but has been detected contaminating a cell culture. Mycoplasma testing (even for unusual species) in cell cultures is essential to ensure the validity and reproducibility of research that uses cell cultures and to ensure the quality of cell line deposits in biobanks. For this reason, it is necessary to perform continuous checks for the absence of Mycoplasma in cell cultures and engage in the continuous adaptation of relevant detection systems.

Keywords: Cell culture, Mycoplasma, Mycoplasma mycoides cluster, PCR, Cell line contamination, Biobank

Introduction

Cell line cultures are a valuable tool for a variety of research areas due to their ability to be indefinitely proliferated. They are extensively employed as in vitro models for disease characterization and drug development (Corral-Vazquez et al. 2017). Work with cell line cultures is faced with two significant challenges, namely (i) cell line misidentification (ATCC-ASN-0002 2010) and (ii) cell culture contamination (Nims and Price 2017). The former occurs due to cross-contamination by other cells from a different subject or as a result of mislabelling. The latter issue is related to biological and chemical contaminants that may adversely affect a cell line culture.

One of the challenges a biobank that works with cell lines must overcome is to verify that the cell lines it stores are entirely free from contamination. For the Biobank of the Public Health System of Andalusia (BBSSPA) (part of the National Bank of Cell Lines (BNLC)), it is essential that we test for the absence of contamination by Mycoplasma in the cell lines that are received by the biobank. This is done during the derivation process and before a cell line is deposited into the biobank.

Among biological contaminants, Mycoplasma contamination is currently considered a major problem in cell cultures (Uphoff and Drexler 2014). The genus Mycoplasma—class Mollicutes—includes prokaryotic microorganisms characterized by a low G + C content, small genome size, and lack of a cell wall. Its small size (d = 0.2–0.7 μm) and flexible membrane allow these bacteria to pass through anti-bacteriological filters (d = 0.45 μm) (Atalla et al. 2015; Sethi et al. 2012). Furthermore, a remarkable dependence on their hosts is explained in terms of their restricted metabolic and physiological pathways (Parker et al. 2018). In relation to cell cultures, because high concentrations of Mycoplasma do not cause turbidity and its effects on cell growth may occur unnoticed, the detection of Mycoplasma presents several difficulties (Young et al. 2010). Although a large number of methods are available for Mycoplasma testing, PCR is the most frequently used and most sensitive testing method (Uphoff and Drexler 2014).

The genus Mycoplasma comprises a large number of species, organized in five phylogenetic groups—hominis, pneumoniae, spiroplasma, anaeroplasma, and asteroleplasma—where the mycoplasmas are arranged. In this study, we examined the Mycoplasma mycoides cluster as well as other related Mycoplasma species of the spiroplasma group (Heldtander et al. 1998; Weisburg et al. 1989), where classification of mycoplasmas has always been controversial because there is a limited number of biochemical and physiological properties that differentiate these species (Pettersson et al. 1996). Recently, by considering phylogenetic relationships based on multilocus sequence typing (MLST) studies, it has been observed that the M. mycoides cluster includes five distinct species: (i) M. mycoides subsp. mycoides, (ii) M. capricolum subsp. capricolum, (iii) M. capricolum subsp. capripneumoniae, (iv) M. mycoides subsp. capri, and (v) Mycoplasma leachii (Fischer et al. 2012). However, several other species, including M. putrefaciens, M. cottewii, and M. yeatsii, have been considered to be very close to the M. mycoides cluster according to their 16S rDNA sequences (DaMassa et al. 1992; Manso-Silvan et al. 2007; Pettersson et al. 1996). Usually, all of these mycoplasmas are pathogens related to cattle (specifically goats and sheep) and are commonly isolated from samples taken from the external ear canals of these animals (DaMassa et al. 1994; Lysnyansky et al. 2016; Manso-Silvan et al. 2007).

Of the more than 200 known species of Mycoplasma, only a small number of these (M. arginini, M. fermentans, M. hominis, M. hyorhinis, M. orale, M. pneumoniae, Acholeplasma laidlawii, Spiroplasma citri and Ureaplasma species) account for the majority of cell culture infections by Mycoplasma (Uphoff and Drexler 2014; Kong et al. 2001). The commercial kits used for the detection of Mycoplasma have been tested and guarantee the detection of the most common mycoplasmas. However, the enormous diversity of existing Mycoplasma species, the appearance of new mutations in these species as a consequence of natural evolution, and their ability (including unusual species) to infect cell cultures suggest that there is a need to perform (i) a continuous review of cell line cultures and (ii) continuous adaptation of diagnostic systems.

In the present study, we describe the detection in cell culture and sequencing of a new species of Mycoplasma, or a new variant of species already known but phylogenetically related to the unusual M. mycoides cluster and to cattle-associated mycoplasmas. This new species has been detected contaminating a cell culture.

Materials and methods

Samples

Cell line supernatants at different passages, which had been checked during the routine generation and characterization procedures of the biobank, were used for Mycoplasma detection. The supernatants were stored at − 80 °C until Mycoplasma DNA isolation and amplification were completed.

Mycoplasma detection

The presence of Mycoplasma in cell culture supernatants was tested by real-time PCR with the Venor GeM qEP kit (Minerva Biolabs, Germany, Cat. No. 11-91200), which guarantees the detection of (at least) the 15 most common Mycoplasma species. For the detection procedure, 500 µl of the supernatants were incubated at 95 °C for 10 min to inactivate DNases and to release DNA from any possible mycoplasmas in the sample. The PCR was performed by following the manufacturer’s instructions using a QuantStudio6 PRO thermocycler (Applied Biosystems, USA) with channels for the detection of FAM and HEX.

Mycoplasma 16S rDNA gene amplification

The complete rDNA gene from Mycoplasma was amplified using the universal primers for 16S rDNA (as proposed by Johansson et al. 1998) over different cell passages. Specifically, different combinations of U1-F and U2-F with U3-R, U5-R, U7-R, and U8-R primers were used following the authors’ recommendations for PCR using a thermocycler (Eppendorf EP gradient S, Germany) (Johansson et al. 1998). A series of 515, 597, 1047, and 1193 bp bands were obtained with U1-F/U3-R, U2-F/U5-R, U2-F/U7-R, and U2-F/U8-R combinations, respectively.

Amplified band purification

The amplified fragments were separated in a 2% agarose gel at 60 V, extracted from the agarose, and purified with Buffer QG (Qiagen, Germany, Cat. No. 19063) and QIAquick PCR purification kit (Qiagen, Cat. No. 28104) according to the manufacturers’ instructions. The amplicons were re-suspended in 30 µl of Buffer EB and quantified (A260) with Nanodrop EP spectrophotometer (ThermoFisher, USA).

DNA sequencing

The purified 16S rDNA fragments were used for sequencing reactions with BigDye Terminator v3.2 Cycle Sequencing kit (Applied Biosystems, USA, Cat. No. 4336917) using a Seq Studio Genetic Analyzer (Applied Biosystems, USA). The sequencing reactions were prepared in a final volume of 10 µl, with 20 ng of DNA, 3.2 pmol of primer forward or reverse (U1-4/U3-R, U2-F/U5-R, U2-F/U7-R, and U2-F/U8-R) and 4 µl of BigDye Terminator 3.1 Ready Reaction mix. The PCR reaction was performed by using a thermocycler (Eppendorf EP gradient S) with an initial incubation step at 96 °C for 1′ and 25 cycles (96 °C 10″, 50 °C 10″, and 60 °C 4′). Subsequently, the reactions were purified with BigDye XTerminator Purification Kit (Applied Biosystem, USA, Cat. No. 4376484) following the manufacturer’s instructions and sequenced by capillary electrophoresis using a SeqStudio Genetic Analyzer Cartridge (Applied Biosystem, USA, Cat. No. A32656) and Plate Manager software with Z_BigDye Terminator v3.1 Dye set and MediumSeq Run module.

Sequence assembly and clustering

The 16S rDNA sequences were analysed and assembled with Variant Reporter 3.0 software (Applied Biosystems, USA) to obtain the 16S rDNA consensus sequence. The 16S rDNA homology was checked by using the BLASTn database (Nucleotide BLAST, National Center for Biotechnology Information) to compare the sequenced 16S rDNA with the other 16S ribosomal RNA sequences that are archived in the rRNA/ITS databases. The representative 16S rDNA sequences from the M. mycoides subcluster and related species (M. putrefaciens, M. cottewii, and M. yeatsii) and the consensus 16S rDNA sequence determined in the present study, were assembled using the Muscle algorithm (Edgar 2004).

Finally, an evolutionary analysis was inferred by using the Maximum Likelihood method (Tamura and Nei 1993). Initial tree(s) for the heuristic search were obtained automatically by applying the Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Tamura–Nei model. Once this was done, the topology with a superior log likelihood value was selected. The evolutionary analysis was conducted in MEGA11 (Tamura et al. 2021).

Results

For this study, Venor GeM qEP kit was used to check for the presence of Mycoplasma in cell culture supernatants. Weak amplification signals were detected during the characterization process of a iPSC cell line derived from peripheral blood mononuclear cells (PBMCs) at two initial passages. This cell line was recorded as ‘positive’ according to the kit’s specifications (data not shown). Cells were cultured in Matrigel-coated plastic using the serum-free, defined media E8. Partial sequences were then amplified with the primers recommended by Johansson et al. (1998). These partial sequences were further sequenced and assembled to create a complete sequence of the 16S rDNA gene of the Mycoplasma that was contained in the sample (accession number OP364020).

After checking the homology of the new Mycoplasma species using the BLASTn database, the highest homologies (> 97%) were detected with mycoplasmas very close to to the M. mycoides cluster. Specifically, a homology of 98.43% was detected for M. yeatsii, and 98.37% for M. cottewii, which corresponds to 22 and 23 nucleotide changes with M. yeatsii rDNA and M. cottewii rDNA, respectively.

An evolutionary analysis was performed by using the Maximum Likelihood method and the Tamura–Nei model. The tree with the highest log likelihood (− 2535.37) is shown (Fig. 1). The tree is drawn to scale, with branch lengths measured in terms of the number of substitutions per site. The Mycoplasma isolated in the present study is shown on a separate branch from the rest of the mycoplasmas belonging to the M. mycoides cluster, although it is closer to the species M. cottewii, M. yeatsii, and M. putrefaciens.

Fig. 1.

Fig. 1

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A phylogenetic tree made with the Maximum Likelihood method and the Tamura–Nei model. Representative strains for M. mycoides cluster and related species were selected. The proportion of sites where at least one unambiguous base is present in at least one sequence for each descendent clade is shown next to each internal node in the tree. This analysis involved nine nucleotide sequences. There were a total of 1468 positions in the final dataset. The accession numbers for the different strains are indicated. The unusual species of Mycoplasma described in this study is indicated and deposited with the accession number OP364020.

Discussion

Different studies have estimated that between 20 and 30% of the cell lines used in research are contaminated with Mycoplasma (Nübling et al. 2015; Drexler et al. 2017). Note too, that verifying the absence of contamination in cell lines is mandatory to guarantee the reproducibility of research results and for biobanking purposes. In the present study, the 16S rDNA gene of a (probably) new Mycoplasma species related with other Mycoplasma species very close to the M. mycoides cluster was sequenced in different passages of a cell line deposited in our biobank; specifically, the new Mycoplasma species indicated a degree of homology greater than 98% with M. cottewii and M. yeatsii. Whilst it is a frequent occurrence to come across cultures contaminated with different Mycoplasma species (Uphoff and Drexler 2014), contamination with species belonging to or phylogenetically close to M. mycoides cluster species usually associated with cattle (DaMassa et al. 1992), has been previously described only twice in the literature in association with cell line cultures. Contamination by a similar Mycoplasma sp., but not identical to M. yeatsii and M. cottewii, has been described, probably from contamination from foetal bovine serum (Calcutt et al. 2015; Del Giudice 1998).

Because the M. mycoides cluster is somewhat confusing phylogenically, different phylogenetic analyses have been reported in the literature. Is currently accepted than the M. mycoides cluster includes only five distinct species: (i) M. mycoides subsp. mycoides, (ii) M. capricolum subsp. capricolum, (iii) M. capricolum subsp. capripneumoniae, (iv) M. mycoides subsp. capri, and (v) Mycoplasma leachii (Fischer et al. 2012). However, somewhat different phylogenetic classifications of species related to the M. mycoides cluster and other mycoplasmas related to cattle have previously been performed. Consequently, Manso-Silván et al. (2007) considered five housekeeping genes for their cluster analysis (fusA, glpQ, gyrB, lepA, and rpoB). These authors distinguished between two subclusters, namely the M. mycoides subcluster (M. mycoides subsp. mycoides LC type, M. mycoides subsp. mycoides SC type and M. mycoides subsp. capri) and the M. capricolum subcluster (M. capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7). In these subclusters (but probably more related to the M. capricolum subcluster), the M. cottewii, M. yeatsii and M. putrefaciens strains can be found (Manso-Silvan et al. 2007). On the other hand, Heldtander et al. suggested that M. cottewii, M. yeatsii, and M. putrefaciens are closely related to each other in a different cluster (Heldtander et al. 1998). Although these phylogenetic classifications have subsequently become obsolete (Fischer et al. 2012), we are certain that there is a group of species that includes M. cottewii, M. yeatsii, and M. putrefaciens which is related to the M. mycoides cluster.

The M. mycoides cluster and related mycoplasmas phylogeny is somewhat confusing because it has been demonstrated that evolution within mycoplasmas is unusually rapid (Woese et al. 1984). This group of mycoplasmas are phylogenetically closely related, but they cause different diseases in animals (DaMassa et al. 1992; Pettersson et al. 1996). It is not possible to provide a general rule concerning how many nucleotides must differ between two isolates for two mycoplasmas to be classified as two different species. It has been accepted that if the 16S rRNA sequence identity for two isolates is below a threshold of 97%, then they are likely to represent different species. However, other data must be considered for identities that lie between 97 and 100% in 16S rRNA sequences to confirm the species’ classification (Murray and Stackebrandt 1995). For the Mycoplasma that was isolated and described in this study, a homology of 98.43% was detected for M. yeatsii and 98.37% for M. cottewii. However, according to the phylogenetic study that we conducted, we conclude with some degree of certainty that we have identified a new species of Mycoplasma, although a higher degree of certainty regarding its classification would demand that we perform additional genetic and biochemical studies.

Regarding the origin of the Mycoplasma contamination described above, we suggest that it is probably related to the use of bovine foetal serum, as claimed in previous studies (Calcutt et al. 2015; Del Giudice 1998), although direct contamination from humans who have been in contact with cattle is also a possible source of contamination.

Conclusion

Mycoplasma testing in cell cultures is essential to ensure the validity and reproducibility of research using cell cultures and to ensure the purity of cell lines tested and deposited in biobanks. Cell line contamination with an unusual species of the Mycoplasma genus in conjunction with its high degree of genetic variability and the rapid evolution of the species of this genus, pose a challenge for its correct detection and identification. Not only does the potential for contamination demand a continuous and scrupulous testing regime, but it also demands that we continuously adapt and evolve our detection methodologies if we are to guarantee the absence of any Mycoplasma species in cell cultures.

Acknowledgements

We would like to thank the administrative help of Susana Alonso Fernández during the administrative processing of the necessary reagents used in this research work and her assistance regarding the publication of the results of our research.

Author contributions

Conceptualization: JAC-Á, RA-Q, and PC; methodology: JAC-Á MVS-P, LG and PC; resources: SP; writing: JAC-Á; review and editing: RA-Q, MVS-P, SP, and PC All of the authors have read and approve of the final manuscript.

Funding

This research was funded by Consejería de Salud y Consumo, Junta de Andalucía, by ISCIII Platform Biobans and Biomodels (PT20/00065) and by Programa operativo de empleo juvenil del Sistema Andaluz del Conocimiento (11B212POEV14).

Data availability

The Mycoplasma sequence described in this study is available from the National Center for Biotechnology Information (NCBI) database with access number OP364020.

Declarations

Conflict of interest

The authors have no competing interests to declare that are relevant to the content of this article.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Jose Antonio Carrillo-Ávila, Email: jantonio.carrillo@juntadeandalucia.es.

R. Aguilar-Quesada, Email: rocio.aguilar.quesada@juntadeandalucia.es

G. Ligero, Email: gertrudis.ligero@juntadeandalucia.es

S. Panadero-Fajardo, Email: sonia.panadero@juntadeandalucia.es

M. V. Santos-Pírez, Email: mvictoria.santos@juntadeandalucia.es

P. Catalina, Email: purificacion.catalina@juntadeandalucia.es

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The Mycoplasma sequence described in this study is available from the National Center for Biotechnology Information (NCBI) database with access number OP364020.

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