Journal of Veterinary Science & Medical Diagnosis ISSN: 2325-9590

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Research Article, J Vet Sci Med Diagn Vol: 7 Issue: 2

Detection of Mycoplasma Spp. in Cell Cultures by Genus-Specific Polymerase Chain Reaction Protocol

Claudia F. Lobos1, María A. Martínez2 and Carlos O. Navarro1*

1Animal Preventive Medicine Department, Microbiology Laboratory, Faculty of veterinary sciences and livestock, University of Chile, Chile

2Microbiology Program ICBM, Medicine Faculty, University of Chile, Chile

*Corresponding Author : Carlos O Navarro
Professor of Biochemistry, Faculty of veterinary sciences and livestock, University of Chile, Chile
Tel: + (56 2) 2978 5627
E-mail:
[email protected]; [email protected]

Received: May 05, 2018 Accepted: May 16, 2018 Published: May 21, 2018

Citation: Lobos CF, Martínez MA, Navarro CO (2018) Detection of Mycoplasma Spp. in Cell Cultures by Genus-Specific Polymerase Chain Reaction Protocol. J Vet Sci Med Diagn 7:2. doi: 10.4172/2325-9590.1000256

Abstract

Contamination of cell cultures with Mycoplasma spp. complicates the basic investigation and the development of biological products. The effects of these bacteria on cultivated cells are changes in the metabolism, immunological and biochemical properties, growth, viability, etc. The Mycoplasma spp. infection on cell cultures might
not be detected by visual inspection or common microscopy. Hence, it is important to go through routine periodic evaluations with a highly sensible and highly specific fast method. Regarding the previous statement, this memoir was based on the molecular diagnosis of Mycoplasma spp., by detecting the 16S rRNA gene through the conventional polymerase chain reaction technic, on cell culture samples from different laboratories of the University of Chile and the Institute of Public Health of Chile. The results obtained in positive controls as in negative controls, allowed the validation of this method in the Faculty of Veterinary Sciences and by applying it on suspicious samples from the Institute of Biomedical Sciences of the University of Chile. This finding was verified by alignment of nucleotide sequences using the Clustal Ω and BLAST software, both online freeware, giving a 97% of nucleotide identity percentage respect to Mycoplasma spp. from the GeneBank®. 

Keywords: Cell cultures; Mycoplasma spp.; 16S rRNA gene; PCR

Introduction

Mycoplasmas are widely distributed in nature as parasites of mammals, birds, reptiles, fish, arthropods and plants [1]. The majority are belonging to the normal microbiota of their hosts. However, some are primary pathogens and many are opportunistic [2]. In veterinary medicine they are a problem when generating pneumonia, pleuropneumonia, pleuritis, aerosaculitis, conjunctivitis, vulvovaginitis, mastitis, chondrodystrophy, otitis and erythrodermatitis in both domestic and laboratory animals [1]. Several species of Mycoplasmas have been described as common contaminants of cell cultures, which represents a serious problem, since they cause various alterations in cellular metabolism and deprive the host cell of essential nutrients [3]. The detection of these microorganisms in cell cultures is currently an essential requirement to achieve the quality standards of laboratories [3]. Multiple methods have been used for its detection, this is how Uphoff and Drexler in 2011 raised the Polymerase Chain Reaction (PCR) as the most recommended method for its sensitivity and specificity. The use of PCR not only allows early detection of contamination by Mycoplasmas in cell cultures, but also provides useful data to determine epidemiological and phylogenetic characteristics [4]. In this context, the implementation of this technique as a routine procedure for the evaluation of the quality of cell cultures would also be useful for the diagnosis of these microorganisms in samples of veterinary origin.

Mycoplasmas belong to the Mollicutes class (from the Latin: mollis, soft, cutis, skin), Mycoplasmatales order, Mycoplasmataceae family, Mycoplasma genus and Ureaplasma, with numerous integrating species [5]. They correspond to one of the smallest existing living organisms, both in size (300 to 800 nanometers (nm)) and in the genome (580 kilobases (kb)). They lack a cell wall, sensitive to lipolytic agents, develop in complex artificial media, have a mainly fermentative metabolism and most are facultative anaerobes [1].

It is worth noting its marked tropism by the cytoplasmic membranes of eukaryotic cells, from which they obtain cholesterol that they incorporate into their own cell membrane to give it greater stability. This property makes them ubiquitous, having been isolated in about 120 animal and plant species [3]. Mycoplasmas usually infect specific hosts but, in the case of animals, some species can be found in different hosts. The human barrier of hosts is rarely crossed by Mycoplasmas of animal origin. However, in cell cultures, both human and animal species can be found [2]. Currently, the contamination of cell cultures with bacteria, fungi and yeasts represents a major problem in laboratories. In general, these organisms are easily detected by turbidity of the cell culture and observation under an inverted microscope, but the Mycoplasmas correspond to a class of bacteria that regularly evades detection [6]. Mycoplasmas, unlike other bacteria, grow slowly and do not produce appreciable changes in crops. However, they produce alterations in cellular metabolism, which is detrimental to the processes of research, diagnosis and production. In other words, the contamination of cell cultures by Mollicutes is a frequent occurrence in laboratories, reporting up to 80% of contaminated crops, which results in unreliable experimental tests and unsafe biological products [3]. In most cases the infection originates from contaminated animal serum and its dissemination in the laboratory is through aerosols. The frequency of infection in cell cultures is approximately 30% [2] and its variation depends on the origin and type of cell culture, duration of the evaluation period and the efficiency of the applied procedures [6]. The dominant contaminant species of Mycoplasma have been maintained over the years. M. hyorhinis, M. orale, M. arginini, M. fermentans and Acholeplasma laidlawii constitute 95% of the identified species. Of these, M. orale and M. fermentans are part of the oropharyngeal and human genital commensal microbiota, while the other species have an animal origin [1,3]. These microorganisms usually adhere to the cells, but depending on the species, they can fuse with the host cell and even invade it, generating multiple consequences. First, a modulation of the immune response is described, which is explained by the ability of Mycoplasmas to both stimulate and suppress T and B lymphocytes. The stimulation is mediated by the induction of proinflammatory cytokines IL-1, IL- 2, IL-4, IL-6, TNF-α, INF-α, INF-β and INF-γ that increase the toxicity of macrophages, NK cells, T and B lymphocytes together with the activation of the complement cascade. On the other hand, the suppression is given by the induction of the cytokines IL-10 and IL-13 that suppresses the proliferation of T lymphocytes and the excessive production of proinflammatory cytokines by macrophages. The modification of cell morphology is the result of the binding of adhesins from the Mycoplasma to components of the extracellular matrix of the host cell by generation of signals that produce specific changes in the cytoskeleton. The interference with viral replication is due to INF-α and INF-β induced by many species of Mycoplasmas, which promote the cytotoxic activity of T lymphocytes and NK cells against cells infected by viruses. Chromosomal modifications result from the release of hydrolytic enzymes such as endonucleases, which suggest chromosomal damage. Finally, the interruption in cellular metabolism considers the sum of all the above effects together with the release of cytotoxic components such as hydrogen peroxide in the host cell, causing oxidative damage and the alteration of the functionality of genes produced by the generation of cascades. Signals from the cytoplasmic membrane to the nucleus [2,5].

Among the methods of the detection of Mycoplasmas as cell culture contaminants, fluorescent DNA staining, 4,6-diamidino- 2-phenylindol dihydrochloride (Hoechst stain or DAPI) and immunological techniques, such as direct immunofluorescence (IFD), have been used. These techniques are quick and simple to perform, but in the case of DAPI staining their sensitivity and specificity is limited and in the DFI they require a panel of monoclonal antibodies that are difficult to obtain [3]. Of course, isolation is always the “golden test” to confirm positivity. However, it is technically very complex and slow, since this microorganism has very particular characteristics, being demanding from the nutritional point of view and sensitive to variations in pH, temperature and osmotic pressure. Therefore, optimum conditions should be guaranteed for their development in culture media to maintain or isolate strains [7].

In past years, PCR has been used as a highly sensitive alternative for the detection of these microorganisms in cell cultures and their inputs [3]. This technique has proven to be a very specific and sensitive method that allows the amplification of low amounts of nucleic acids to a level that can be easily detected [4].

Given the large number of species that can be detected as contaminants, the most used primers are designed to recognize the 16S rRNA gene [8], considering its constitutive and highly conserved character [9].

The 16S rRNA corresponds to a polyribonucleotide encoded by the rrs gene or ribosomal DNA (16S rDNA) and included in the 30S subunit of the bacterial ribosome. Its sequence presents approximately 1500 base pairs (bp) and is composed of speciesspecific variable zones and conserved zones [10]. The same authors point out that the chosen universal splitters are complementary to the conserved zones of the start and end of the gene. On the other hand, the variable zones comprised between these conserved zones are the regions used to perform a comparative taxonomy. Currently, GenBank® is the database with more information since it contains more than two million deposited sequences of this gene, however, those relevant are available for only a limited number of Mollicutes. Some years ago, a specific PCR protocol for the genus Mycoplasma was developed using the specific reverse splitter MGSO [11] in combination with a new splitter called GPF, to amplify a larger PCR product, resulting in a fragment of 1013 bp. The design of GPF was made based on the comparison and alignment to the 16S rRNA gene of numerous known Mycoplasma species with the use of the Clustal W program [12].

The specificity of this PCR was confirmed using 35 different species of Mycoplasma and 9 other bacteria with a cell wall as reference [12]. As a result, a DNA fragment of the expected size was amplified in 100% of the tested Mycoplasma isolates and in none of the other bacteria. Finally, its sensitivity was evaluated by means of 10 serial dilutions of M. synoviae and M. gypis DNA, as well as cultures of these same species, delivering a value of 1 pg of DNA (1 CFU).

In general terms, the same authors describe the genus-specific PCR for Mycoplasma as a good diagnostic tool because there was no risk of contamination, the modification of the initial technique did not reduce its sensitivity and the size of the amplified product allowed differentiation by species of this bacterium through its sequence. According to this background, a PCR protocol aimed at detecting the 16S rRNA gene was developed in this title report since it is considered a useful procedure for the diagnosis of these microorganisms in cell cultures. However, although the detection of a 1013 bp fragment is necessary, it is not sufficient to guarantee the presence of Mycoplasma spp. in a sample. Therefore, the next stage contemplated the sequencing of the amplified and the incorporation of this sequence in a freely available on-line software called BLAST (Basic Local Alignment Search Tool) to obtain a Nucleotide Identity Percentage (NIP) respect to the Genbank®. In this way, if two genes are considered different from each other compared to a sequence identity value less than or equal to 79% [10], a PIN value of the samples of this study greater than 80% guaranteed the presence of Mycoplasma spp.

In synthesis, a constitutive fragment of the 16S rRNA gene of Mycoplasma spp. validated by the nucleotide sequencing carried out was detected by genre-specific PCR. In this way, a rapid and effective routine procedure was established to detect early contamination of this bacterium, thus avoiding future problems related to scientific work both in the laboratories of the Departments of Animal Preventative Medicine and Animal Pathology of the Faculty of Medicine. Veterinary Sciences and Cattle of the University of Chile, as in other dependencies that require it.

Materials and Methods

Samples and controls

This study was conducted in the Microbiology and Virology laboratories of the Department of Animal Preventive Medicine, Faculty of Veterinary and Animal Sciences (FAVET), University of Chile. For the implementation of the PCR, DNA of M. pneumoniae strain FH from the Academic Unit of Parasitology of the Institute of Biomedical Sciences (ICBM), University of Chile was used as a positive control, and bovine serum-free culture media were used as negative controls, DNA of viral and bacterial origin (Canine herpesvirus type 1, Staphylococcus intermedius and Enterococcus faecium) maintained at -20ºC in the Microbiology and Virology laboratories of FAVET, Universidad de Chile. Additionally, nuclease-free water was used as reagent control.

Once the PCR protocol was established, 20 samples from positive cell cultures (Virology Laboratory (ICBM), Academic Parasitology Unit (ICBM), Virology Unit of the Institute of Public Health (ISP) and 20 samples from of suspicious samples (Fish Pathology and Virology Laboratories, FAVET) of infection by Mycoplasma spp. The extraction of the bacterial DNA from the cell cultures an aliquot of 500 μL of the supernatant of each, using the commercial kit Wizard SV Genomic DNA® (Madison, USA), according to the manufacturer’s instructions The amount of DNA obtained was quantified by absorbance measurement at 260 nm [5] in a spectrophotometer (UNICAM UV/vis®) from the FAVET Biochemistry Laboratory To verify possible differences in the detection by PCR, each suspicious sample was studied with and without extraction of the corresponding DNA. As negative controls, the use of 20 samples from cell culture media free of fetal bovine serum and of routine use was contemplated.

Detection of the 16S rRNA gene of Mycoplasma spp. by genus-specific PCR

Primers: In the PCR reaction, the primers GPF 5’-GCTGGCTGT GTGCCT-3’ and MGSO 5’ TGCACCATCTGTCACTCTGGTACCCTC-3’ were used to generate a fragment of 1013 bp [7]. Both primers were synthesized by the Center Technological Support Equipment and Services of the ICBM, University of Chile.

Mixture of the reaction (in triplicate): To achieve the amplification mixture of the purified DNA, a 2X PCR Master Mix Fermentas® kit was used, which includes the thermostable polymerase, the deoxynucleotide triphosphates (dNTPs), the reaction buffer and MgCl2, that 15 μL were extracted and poured into a 0.2 mL Eppendorf tube, together with 5 μL of each of the primers, and 5 μL of the template DNA sample, obtaining a volume total of 30 μL.

DNA amplification: The protocol was defined by an initial incubation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 30 s, alignment at 56°C for 30 s and synthesis at 72°C for 30 s. Once the cycles were completed, a final elongation was performed at 72°C for 10 min [12].

Visualization of the amplified product: It was performed by electrophoresis in 2% agarose gel (Winkler®) in Tris Acetate EDTA (TAE) buffer (Fermentas®), which was then subjected to electrophoresis by immersion in ethidium bromide (0.5 μg/mL) (Fermelo®). The PCR product was mixed with 6 μL of the commercial loading product, 6X “Mass Ruler Loading Dye Solution” (Fermentas®), which has glycerol to give density to the sample and bromophenol blue to check the progress of the migration of the DNA bands. An aliquot of 5 μL of this mixture was deposited in the respective well of the gel. Electrophoresis was carried out at 90 V for 90 min. 5 μL of HyperladderTM was used IV (Bioline®) as molecular size marker (MTM), which contains DNA fragments between 100 and 1,000 bp to facilitate the detection of amplified fragments. After the procedure, the gel was incubated in ethidium bromide (0.5 μl /ml) for 30 min and once stained it was visualized in the ultraviolet transilluminator (Transiluminator UVP®), to finally be photographed with a digital camera.

Determination of the nucleotide identity percentage (NIP) respect to GenBank®

Sequencing: DNA fragments obtained by PCR were sent to the company Genytec for the determination of its nucleotide sequence.

Analysis: The obtained sequences were aligned using the Clustal W 2.2.012 program initially to obtain a consensus sequence and then the program was used BLAST to establish the NIP respect to official sequences of the 16S rRNA gene available in the GenBank®.

Analysis of results: Every sample that in the visualization under UV light originated a fragment of DNA close to 1000 bp and NIP was greater than or equal to 80% (NIP ≥ 80%) was considered suspicious to come from Mycoplasma spp.

Biosafety: The measures associated with this study consisted of limited access to the facilities, use of a biosafety cabinet at the time of bacterial DNA extraction, use of Bunsen burner to delimit a bioclean work area and apron use. It should be noted that both positive and negative controls corresponded to DNA previously extracted and stored at -20°C. In the case of cell culture samples, the Eppendorf tubes that contained them were subjected to 80°C for ten minutes before processing them. For the realization of the PCR, it was required to delimit a clean and exclusive zone to avoid contamination with genetic material not coming from the sample and the use of latex gloves to carry out the procedures. For the visualization of the PCR product it was necessary to use gloves, because ethidium bromide was used, which has mutagenic properties. At the time of using the ultraviolet light transilluminator, it was required to have glasses with UV filter and an acrylic plate placed between the equipment and the operator. Finally, the gel was incinerated in FAVET together with the gloves that were used for its handling.

Results

Genus-specific PCR of Mycoplasma spp.

When PCR was carried out in 20 DNA samples of positive cell cultures, as well as in 20 samples of different origin (viral and bacterial) and cell culture media free of fetal bovine serum, it was possible to verify their status. For example, Figure 1 show the result obtained by four positive samples and four negative samples. Thus, after the incubation of the gel in ethidium bromide, in the wells that included positive samples, a DNA fragment of a size close to 1000 bp was visualized, represented by a single band, sharp and wide. When applying this methodology to the 20 DNA samples suspected of Mycoplasma spp. infection, only one of them was positive and, as an example, is plotted in Figure 2 next to the positive control. This sample, from a culture supernatant of MDCK cells, was positive with and without DNA extraction, being observed in both cases a fragment of DNA of molecular size about 1000 bp and equal display quality.

Figure 1: Detection of the 16S rRNA gene of Mycoplasma spp. by genrespecific PCR. Electrophoresis in 2% agarose gel. Lane 1: MTM (100-1,000 bp); Lane 2-5: positive samples; Lane 6-9: negative samples; Track 10: MTM (100-1000 bp); MT: Molecular size marker: HyperladderTM IV (Bioline®).

Figure 2: Detection of the 16S rRNA gene of Mycoplasma spp. by genrespecific PCR. Electrophoresis in 2% agarose gel. Lane 1: negative sample1; Lane 2: negative sample2; Lane 3: negative sample1; Lane 4: negative sample2; Lane 5: positive sample1; Lane 6: positive sample2; Lane 7: positive control (M. pneumoniae strain FH); Lane 8: negative control (HCV-1); Lane 9: negative control (S. intermedius); Lane 10: MTM (100-1000 bp, Bioline®); (1) with DNA extraction, (2) without DNA extraction.

Analysis of the sequenced DNA fragment

The amplified from the positive sample were sent in quintuplicate to sequencing Genytec Ltda. and five sequences of around 500 bp were received (Figure 3).

Figure 3: Sequences obtained from Genytec Ltd.

Then, this sequence was analyzed according to the BLAST program (Figure 4a and Figure 4b) which determined a NIP of 97% respect to the 16S rRNA gene of Mycoplasma spp.4

Figure 4a: The CFLF consensus sequence obtained by Clustal Ω.

Figure 4b: Nucleotide identity percentage by Blast for the CFLF consensus sequence.

Discussion

The amplification products were characterized by their great intensity and sharpness, not observing bands of non-specific amplification or DNA degradation in any case. It should be mentioned that 100% of the positive control samples generated a single band close to 1000 bp, consistent with the size of the 16S rRNA gene. On the other hand, the high NIP obtained shows that indeed the DNA fragment amplified in the only positive suspect sample would correspond to the 16S rRNA gene of Mycoplasma spp. Said positive sample of the contamination of Mycoplasma spp. with and without extraction of DNA, corresponds to a cell culture of MDCK cells that presented problems in its growth in the Virology Laboratory of FAVET. This situation was reported to the provider (ICBM), which confirmed his status. The bands generated in the electrophoresis by said sample, both from the direct culture and from the purified DNA, showed a similar degree of fluorescence and suggest that in both cases it was possible to detect a similar amount of DNA in the amplicons. The above, according to the comparison of its fluorescence with respect to the MTM, which not only provides band size information but also gives an approximation about the amount of DNA.

The high NIP obtained to indicate the presence of Mycoplasma spp. However, the BLAST program would indicate that it is M. hyorhinis or M. hyopneumoniae. For the identification of the Mycoplasma species, it would be necessary to sequence a greater number of bases of sequence more than 500 bases, which is the maximum that the Genytec team sequenced. However, this has not impact on the effects of this Work, since the objectives have been met with great success.

It should be noted, in relation to the information provided by the BLAST software, that M. hyorhinis and M. hyopneumoniae correspond to one of the most commonly identified contaminating species [1,3]. Based on this, it is inferred that the contamination of the only affected cell culture would be the product of the use of animal serum infected with the bacteria [13]. For this reason, it is necessary to use specific control measures, such as genus-specific PCR used in this study.

In this work it was evidenced that the detection of Mycoplasma spp. by genus-specific PCR it would not require the purification of bacterial DNA from the supernatant of the cell culture, but it can be performed directly with the previous condition of submitting the sample at 80ºC for ten minutes to avoid the risk of infection. This, in addition to influencing the time and costs involved, avoids the possibility of obtaining false negatives by the action of PCR inhibitors. A wide range of substances, which are mostly part of the sample and others added to the reaction mixture, can strongly inhibit the activity of the polymerase, thus limiting the use of the PCR technique. These inhibitors include reagents used during the DNA extraction and purification step, such as phenol and ionic detergents that can denature said enzyme [14,15]. The above, together with the error rate of the enzyme Taq polymerase, justifies the sequencing of the positive sample in fivefold that was done in this investigation. Finally, those possible errors are minimized by aligning the results of the sequencing and generating a consensus sequence.

The general objective of this report was to implement the molecular diagnosis of Mycoplasma spp. in experimental cell cultures by PCR in our laboratory. In relation to this technique, nested PCR (n-PCR) is described as a modification of the PCR and is used to increase the sensitivity of the assay in up to 10 orders with respect to conventional PCR. In a typical protocol for an n-PCR, a first round of PCR is executed with a pair of external primers. Then, a small amount of the product of this first round is transferred to a new reaction tube for a second round in which a pair of internal primers is used. The decrease in the number of false negatives that is achieved with the use of n-PCR, gives greater reliability to the monitoring method and reduces the possibilities of making errors in the assessment of the results [4]. However, the foregoing does not threaten the genus-specific PCR used in this study, since in the n-PCR there is a substantial risk of cross-contamination by the products of the first round of amplification they are used in the second round, resulting in the transfer of material between the different PCR tubes [16,17].

Also related to contamination in laboratories, once a cell culture infected with Mycoplasma spp. is detected, its immediate elimination is recommended. However, those with special research value can be treated [2]. The efficacies of the elimination of Mycoplasmas from contaminated cell cultures vary from 71-86%, according to the antimicrobial scheme used. However, the overall effectiveness of decontamination using more than one scheme if there is a failure in the eradication of microorganisms against a treatment is 96%. Failures of eradicating Mycoplasmas are due to antimicrobial resistance (3- 20%) or to the cytotoxic effect of the antimicrobial used (3-11%). The most commonly used antimicrobials correspond to tetracyclines, quinolones and macrolides, taking into consideration that sensitivity to different macrolides differs between Mollicutes species [4]. The foregoing makes the sequencing stage essential based on their differentiation by species in the affected cell cultures. Finally, the 6 weeks of freezing at -20°C of the control samples did not influence the detection of Mycoplasma spp. by PCR, since all positive and negative cell cultures processed resulted as such.

Conclusions

The amplification of the 16S rRNA gene was adequate for the diagnosis of Mycoplasmas in cell cultures. The NIP obtained from 97% indicates that the test is specific for the detection of Mycoplasma spp. This diagnostic technique had the same results, either using DNA extraction or applying it without this procedure, which offers a faster, cheaper and simpler variant of this Mycoplasma detection methodology.

References

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