Differentiation of Mycoplasma Species by 16S Ribosomal DNA PCR and Denaturing Gradient Gel Electrophoresis Fingerprinting

Abstract

Denaturing gradient gel electrophoresis (DGGE) of a 16S ribosomal DNA PCR product was used to differentiate 32 mycoplasma species of veterinary significance. Twenty-seven (85%) species could be differentiated by DGGE. This method could enable the rapid identification of many mycoplasma species for which there is no specific PCR available and which are currently identified by using culture and serological tests.

Mycoplasmas belong to the class Mollicutes and are among the smallest free-living microorganisms capable of autoreplication. Mycoplasmas are highly fastidious bacteria, difficult to culture and slow growing. Many species are important veterinary pathogens causing respiratory infection, mastitis, conjunctivitis, arthritis, and occasionally abortion; they include Mycoplasma mycoides subsp. mycoides small colony (SC), the causative agent of the Office International des Epizooties list A disease contagious bovine pleuropneumonia (12). Identification of mycoplasmas as the causative agents of disease is often hindered by the lack of rapid diagnostic tests together with similarities in the clinical diseases that they cause. Conventional methods of diagnosis are based on culture and serological tests, such as the complement fixation test (9), enzyme-linked immunosorbent assay (2), and immunoblotting (15), and can be time-consuming, insensitive, and nonspecific.

Recently, PCR has been employed for the laboratory diagnosis of some veterinary mycoplasmas, including species of the closely related M. mycoides cluster (3), Mycoplasma gallisepticum (5), Mycoplasma hyopneumoniae (19), and Mycoplasma bovis (1). Differentiation of mycoplasma by PCR based on specific primers has been limited, as there is little interspecific variation in 16S ribosomal DNA (rDNA). The identification of alternative genes suitable for PCR has been hampered by the lack of sequenced animal mycoplasma genomes. To date, there has not been a single generic test that enables mycoplasma identification to the species level.

Denaturing gradient gel electrophoresis (DGGE) can theoretically detect single base mutations in DNA (4, 7). The method is based on the prevention of migration of DNA fragments following strand separation caused by chemical denaturants. DGGE has been used extensively for diversity analysis in microbial ecology (10) but has not been widely used for the identification and differentiation of pathogenic bacteria except for the detection and identification of Listeria species (N. Schmolker, F. van Ommen Kloeke, and G. Geesey, Bio-Rad Mutation Analysis, technical note 2403) and for the molecular typing of Staphylococcus aureus (6) and Campylobacter species (16). This study used PCR-DGGE of the V3 region of the 16S rDNA gene to differentiate 32 species of mycoplasma commonly associated with animal disease.

The mycoplasma strains used in this study are listed in Table 1. All strains were cultured as described previously (13). Mycoplasma DNA was extracted from a 1-ml aliquot of stationary-phase culture as described previously (17). Amplification of the V3 region of the 16S RNA gene was performed according to the method of Muyzer et al. (11) with universal bacterial primers GC-341F (5′-CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG GCA CGG GGG GCC TAC GGG AGG CAG CAG) and 534R (5′-ATT ACC GCG GCT GCT GG) (11). For PCR, 1 μl of lysate was added as a template to 49 μl of a reaction mixture containing 10 mM Tris-HCl (pH 9.0), 1.5 mM MgCl₂, 50 mM KCl, 0.1% Triton X-100, 0.2 mM (each) deoxynucleoside triphosphates, and 0.5 U of Taqgold (Applied Biosystems). The cycling conditions were: denaturation at 94°C for 5 min followed by 30 cycles of 95°C for 1 min, 55°C for 45 s, and 72°C for 1 min and a final extension step of 72°C for 10 min, and samples were kept at 4°C until analysis. DGGE was performed with the Ingeny PhorU 2 × 2 apparatus (GRI Molecular Biology, Essex, United Kingdom). Samples were loaded onto 10% polyacrylamide-bis (37.5:1) gels with denaturing gradients from 30 to 60% (where 100% is 7 M urea and 40% [vol/vol] deionized formamide) in 1× Tris-acetate-EDTA electrophoresis buffer (Severn Biotech Ltd., Worcestershire, United Kingdom). Electrophoresis was performed at 100 V and 60°C for 18 h. Gels were then stained with SBYR Gold (Cambridge BioScience, Cambridgeshire, United Kingdom) in 1× Tris-acetate-EDTA for 30 min at room temperature and visualized under UV illumination.

TABLE 1.

Mycoplasma species used in this study

Species	Strain	Host
M. cloacale	NCTC 10199	Avian
M. gallinarum	NCTC 10120	Avian
M. gallisepticum	NCTC 10115	Avian
M. gallopavonis	NCTC 10186	Avian
M. glycophilum	NCTC 10194	Avian
M. iners	NCTC 10165	Avian
M. iowae	NCTC 10185	Avian
M. lipofaciens	NCTC 10191	Avian
M. meleagridis	NCTC 10153	Avian
M. synoviae	NCTC 10124	Avian
M. agalactiae	NCTC 10123	Ovine/caprine
M. arginini	NCTC 10129	Ovine/bovine
M. capricolum subsp. capricolum	NCTC 10154	Caprine
M. capricolum subsp. capripneumoniae	NCTC 10192	Caprine
M. conjunctivae	NCTC 10147	Ovine
M. cottewii	NCTC 11732	Caprine
M. mycoides subsp. mycoides LC	F30	Ovine/caprine
M. mycoides subsp. capri	NCTC 10137	Caprine
Mycoplasma ovine/caprine serogroup 11	2D	Ovine/caprine
M. ovipneumoniae	NCTC 10151	Ovine
M. putrefaciens	NCTC 10155	Ovine/caprine
M. flocculare	NCTC 10143	Porcine
M. hyopneumoniae	NCTC 10110	Porcine
M. hyorhinis	NCTC 10130	Porcine
M. hyosynoviae	NCTC 10167	Porcine
M. bovigenitalium	NCTC 10122	Bovine
M. bovirhinis	NCTC 10118	Bovine
M. bovis	NCTC 10131	Bovine
M. bovoculi	NCTC 10141	Bovine
M. canis	PG14	Bovine/canine
M. californicum	NCTC 10146	Bovine/canine
M. dispar	NCTC 10125	Bovine

PCR products of approximately 340 bp were obtained for all 32 Mycoplasma species tested. These products were subjected to DGGE in groups according to host animal. In the majority of cases only one band was seen, indicating that there was no intraspecific variation in the amplified sequence. The presence of multiple bands indicated that more than one 16S rDNA operon was present and that there were some sequence differences between the copies.

The use of DGGE to separate PCR products according to differences in primary sequence enabled differentiation of all 10 avian mycoplasmas species analyzed (Fig. 1). This indicated that the sequence was different for each isolate. However, DGGE could differentiate only 7 of the 10 small-ruminant mycoplasmas tested, which was due to identical DGGE profiles being obtained for the closely related members of the M. mycoides cluster Mycoplasma capricolum subsp. capripneumoniae, Mycoplasma mycoides subsp. capri, M. mycoides subsp. mycoides large colony, and Mycoplasma putrefaciens (Fig. 2). Interestingly, M. putrefaciens also gave a profile identical to that of the M. mycoides cluster members, providing further evidence that M. putrefaciens should be included in the M. mycoides cluster (21). Although DGGE was unable to distinguish many of the M. mycoides cluster members when PCR of the 16S gene was used, it is possible that DGGE based on alternative targets, such as the 16S-23S intergenic spacer region, might enable differentiation.

FIG. 1.

DGGE of avian mycoplasmas. Lanes: 1 and 12, Escherichia coli marker; 2, M. gallisepticum; 3, M. gallinarum; 4, M. iners; 5, M. gallopavonis; 6, M. lipofaciens; 7, M. cloacale; 8, M. iowae; 9, M. glycophilum; 10, M. synoviae; 11, M. meleagridis.

FIG. 2.

DGGE of ovine and caprine mycoplasmas. Lanes: 1 and 13, E. coli marker; 2, M. agalactiae; 3, M. ovipneumoniae; 4, M. ovine/caprine serogroup 11; 5, M. cottewii; 6, M. capricolum subsp. capripneumoniae; 7, M. arginini; 8, M. conjunctivae; 9, M. mycoides subsp. capri; 10, M. capricolum subsp. capricolum; 11, M. mycoides subsp. mycoides large colony; 12, M. putrefaciens.

All four porcine mycoplasmas gave different DGGE profiles (Fig. 3). DGGE also enabled the differentiation of five bovine mycoplasmas (Fig. 4). However, Mycoplasma californicum and Mycoplasma dispar could not be distinguished, and the banding patterns of M. bovis and Mycoplasma canis were similar. Interestingly, the migration of the DGGE band obtained for the bovine pathogen Mycoplasma bovigenitalium was identical to the small-ruminant mycoplasma Mycoplasma ovine/caprine serogroup 11 (Fig. 5). These results are not surprising, as it has recently been proposed that Mycoplasma ovine/caprine serogroup 11 and M. bovigenitalium should be redesignated as the same species based on their close phenotypic and genotypic similarity (14).

FIG. 3.

DGGE of porcine mycoplasmas. Lanes: 1 and 6, E. coli marker; 2, M. flocculare; 3, M. hyopneumoniae; 4, M hyosynoviae; 5, M. hyorhinis.

FIG. 4.

DGGE of bovine mycoplasmas. Lanes: 1 and 9, E. coli marker; 2, M. bovis; 3, M. bovoculi; 4, M. bovirhinis; 5, M. californicum; 6, M. canis; 7, M. bovigenitalium; 8, M. dispar.

FIG. 5.

DGGE of M. bovigenitalium and Mycoplasma ovine/caprine serogroup 11. Lanes: 1 and 4, E. coli marker; 2, M. bovigenitalium; 3, Mycoplasma ovine/caprine serogroup 11.

To determine the robustness of DGGE for mycoplasma species identification, 20 strains of M. mycoides subsp. mycoides SC from Africa, Australia, and Europe were compared by using DGGE. The same DGGE profile was obtained with 22 of the 23 M. mycoides subsp. mycoides SC strains tested. The only strain that gave a different DGGE profile was a vaccine strain, T₁44 (results not shown). Previous studies have also indicated that the vaccine strains of M. mycoides subsp. mycoides SC are genotypically different from the field strains (8, 20). Clearly, further work on more strains of each species needs to be carried out in order to confirm that the same profiles are obtained.

This is the first study to detect and differentiate all major mycoplasmas of veterinary importance with a single test. DGGE of the 16S rRNA gene could differentiate almost all mycoplasmas within a host animal group. The application of this technique could be particularly useful for mycoplasmas that are difficult to culture, such as porcine mycoplasmas. In addition, mycoplasmas that are difficult to distinguish by using normal culture and serological techniques, including M. bovis and Mycoplasma agalactiae, can be differentiated (13).

As universal bacterial primers were used, the approach described here will not be limited to species that have already been characterized; novel and unknown species will also be detected. The disadvantage of this is that bacteria other than members of the Mollicutes will also generate a band on the DGGE gel, which may give confusing results unless the Mycoplasma organisms have been enriched by cultivation. This may be overcome by designing Mycoplasma-specific primers.

Although DGGE is rapid compared with traditional culture and serological techniques (typically taking less than 24 h compared with up to 2 weeks for serological or culture-based diagnosis), it is feasible that the process could be accelerated further by the use of DNA chip technology. The use of 16S oligonucleotide probes has already been applied to the detection of soil microorganisms (18), and there is increasing interest in its use for the detection of pathogenic bacteria (22).