Herein, we present data demonstrating that the application of initial cell culture enrichment could significantly improve mycoplasma testing methods based on the nucleic acid amplification technology (NAT) including a polymerase chain reaction (PCR)/microarray method. The results of the study using Vero cells demonstrated that this cell culture is able (1) to support efficient growth of mycoplasmas of primary interest, i.e., species found to be cell line contaminants, (2) to increase the sensitivity of NAT assay to the detection limits of the conventional broth/agar culture methods, and (3) to reduce the time required for mycoplasma testing fourfold in comparison with the conventional methods. Detection and identification of mycoplasmal agents were conducted using a modified PCR/microarray assay based on genetic differences among Mollicutes in the 16S-23S rRNA intergenic transcribed spacer (ITS). The application of nano-gold/silver enhancement technology instead of previously used fluorescent dyes significantly simplified the readout of microarray results and allowed us to avoid using expensive scanning equipment. This modification has the potential to expand the implementation of microarray techniques into laboratories involved in diagnostic testing of mycoplasma contamination in cell substrates and potentially in other biological and pharmaceutical products.

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Cell extracts of the fermentative Mollicutes Acholeplasma laidlawii B-PG9, Acholeplasma morum S2, Mycoplasma capricolum 14, Mycoplasma gallisepticum S6, Mycoplasma pneumoniae FH, Mycoplasma hyopneumoniae J and M. genitalium G-37, and the non-fermentative Mycoplasma hominis PG-21, Mycoplasma hominis 1620 and Mycoplasma bovigenitalium PG-11 were examined for 39 cytoplasmic enzyme activities associated with the tricarboxylic acid (TCA) cycle, transamination, anaplerotic reactions and other enzyme activities at the pyruvate locus. Malate dehydrogenase (EC 4.2.1.2) was the only TCA-cycle-associated enzyme activity detected and it was found only in the eight Mycoplasma species. Aspartate aminotransferase (EC 2.6.1.1) activity was detected in all Mollicutes tested except M. gallisepticum S6. Malate synthetase (EC 4.1.3.2) activity, in the direction of malate formation, was found in the eight Mycoplasma species, but not in any of the Acholeplasma species. Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was detected in the direction of oxaloacetate (OAA) formation in both Acholeplasma species, but not in any of the Mycoplasma species. Pyruvate carboxylase (EC 6.4.1.1), pyruvate kinase (EC 2.7.1.40), pyruvate dehydrogenase (EC 1.2.4.1) and lactate dehydrogenase (EC 1.1.1.27) activities were found in all ten Mollicutes tested. No activities were detected in any of the ten Mollicutes for aspartase (EC 4.3.1.1), malic enzyme (EC 1.1.1.40), PEP carboxytransphosphorylase (EC 4.1.1.38), PEP carboxykinase (EC 4.1.1.32) or pyruvate orthophosphate dikinase (EC 2.7.9.1). In these TCA-cycle-deficient Mollicutes the pyruvate-OAA locus may be a point of linkage for the carbons of glycolysis, lipid synthesis, nucleic acid synthesis and certain amino acids. CO2 fixation appears obligatory in the Acholeplasma species and either CO2 fixation or malate synthesis appears obligatory in the Mycoplasma species.

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