Iranian Journal of Medical Microbiology

Introduction
Mycoplasmas are microorganisms which lack cell walls, but they have particular sterol plasma membranes (1,2). They are distinctive from other bacteria taxonomically and are in Mollicutes class (15). These days, research laboratories use the cells and cell culture techniques for regenerative medicine (3, 4) and biotechnological productions (1,7). After establishment of cell culture laboratories, contamination of cell cultures with Mycoplasmas have occurred. So, one of the problems in the cell culture is that laboratories could spread Mycoplasmas in all cell cultures, and it could promote between the other cell cultures (5,7). Using unspecific methods to identify Mycoplasmas in cell cultures is not so reliable because Mycoplasmas may not have any cytopathic effect or turbidity in some culture cell lines. Whereas Mycoplasmas could contaminate the cell lines which they are used in manufacturing bioproducts such as vaccination so detecting of these microorganisms in cell lines have been so important and valuable (5,16). Some of the various species of Mycoplasma which could contaminate cell cultures  are Mycoplasma hyorhinis, Acholeplasma laidlawii Mycoplasma pneumoniae (M. pneumoniae), Mycoplasma arginini, Mycoplasma orale and Mycoplasma fermentans (5,12,16).  M. pneumoniae is one of the small microorganisms which could live free in nature. Its size is about 125-250 nm and it grows in cell-free media not so fast. It may be necessary to incubate diphasic media for 2-3 weeks with periodic subcultures onto serum agar plates before an isolate is obtained Furthermore, it appears that the widely used Hayflick medium may not be optimal for the cultivation of all strains of M. pneumoniae.
 
Materials and Methods
Mycoplasma culture
For M. pneumoniae isolation, 82 cell culture samples were collected and cultured in PPLO broth media supplemented. All these samples were analyzed by culture and polymerase chain reaction (PCR) to detect M. pneumoniae. Overall, 82 samples were transferred to Mycoplasma reference laboratory on ice packs. Primarily, the isolated samples were passaged and filtered in fresh PPLO broth then inoculated on PPLO agar medium (BBL, Becton Dickinson and company, Cockeyville, Sparks, MD, USA). Both the broth and agar cultures were incubated at 37 °C in 50% Co₂ and 98% humidity (18,20).
DNA Extraction and PCR.
In this study we used a previously described method, with some modifications, for the extraction of DNA by Kojima et al. (18). From each of cell culture samples, 0.5 mL was transferred to Eppendorf tube and centrifuged for 15 min at 13000 rpm. The supernatant fluid was discarded, and the lysis buffer was added to the tube and incubated for at least 4 hours at 56ºC. After that, phenol, phenol, chloroform (1:1) and chloroform were added to the tube individually and respectively and mixed them. Then in each step the tubes were centrifuged at 13000 rpm for 15 min and aqueous layer (top layer) was carefully transferred into a new tube for the following steps. Eventually sodium acetate and ethanol (ETOH) were added in the tubes and mixed well. The tubes were then frizzed in -20ºC for 20 minute and centrifuged for 15 min at 13000 rpm. Finally, 200 µL of 70% ETOH was added and the tubes were centrifuged for 5 min at 13000 rpm. The supernatant was discarded, and the pellet was dried after which 50 µL of distilled water was added to the tubes.
The PCR detection of Mycoplasma genus was performed (18), after that each sample were positive in genus Mycoplasma PCR, were analyzed by the PCR  detection of M. pneumoniae in 543bp fragment by using of MPF and MPR primers which obtained from P1 adhesin gene (10).
 
Results   
Out of the 82 cell cultures samples, 31(37.8%) were positive for Mycoplasma culture, and showing typical Mycoplasma colonies grown on PPLO agar. In addition, 48 (58.5%) samples had positive PCR results for Mycoplasma genus (Figure 1) among which no M. pneumoniae was found (Figure 2) (Tables 1 and 2).

    Table 1. Primers for detection of Mycoplasma in genus PCR

             Table 2. Primers for detection of M. pneumoniae in PCR

              Table 3. Consequences of culture and PCR surveys for detecting of Mycoplasma genus

    Table 4. Consequences of culture and PCR surveys for detecting of M. pneumoniae

M. pneumoniae is one of microorganisms which could contaminate of the cell cultures. Whereas cell cultures which could be used in vaccination, so detection of this pathogen from cell cultures could be important. In the present study M. pneumoniae was not isolated and detected from cell culture samples by using culture and PCR assay.
Robinson and coworkers in 1956 detected Mycoplasma in cell culture for the first time when they tried to investigate the consequences of PPLO on Hela cells (24). Mycoplasmas are the contamination of the cell cultures; also, they could change cell cultures’ function, growth, metabolism, morphology. Spread and extension of this microorganism among the cell cultures could damage the cell cultures, change their functions of each cell cultures and create CPE with plaque formation in them (5,12,16). The present study showed that 37.8% of the culture samples were positive for presence of Mycoplasma in culture method and 58.5% of the samples were positive in PCR assay. So according to results of this study PCR method could be more reliable than the traditional methods such as culture for detecting M. pneumoniae in cell culture therefore PCR assay could be an alternative method for detection of M. pneumoniae. It is also recommended to use the novel molecular methods such as Real-Time PCR for the detection of Mycoplasmas in cell cultures since these methods are fast, sensitive, specific and low cost. Also, this study showed that there was no contamination of M. pneumoniae among the cell culture samples referred to the Mycoplasma Reference Laboratory of Razi Institute.