Biomolecular Characterization of Trichomonas vaginalis from Spain: Evaluating Genetic Correlation with Drug Resistance and Endobionts

Abstract

Trichomonas vaginalis causes one of the most prevalent sexually transmitted infections (STIs). Despite extensive biomolecular research on this protozoan, no efficient molecular tool currently exists for the intraspecific classification of T. vaginalis isolates. In recent years, an incipient tendency has been observed to classify this parasite into two genotypes that correlate in a high percentage with phenotypic characteristics (such as metronidazole resistance and viral endobionts). However, this grouping method has not yet been consolidated by the scientific community. In this sense, Mycoplasma hominis has also been identified as an endobiont of T. vaginalis, but no previous studies on its presence and possible correlation with the two T. vaginalis populations detected have been executed. In the present study, several new T. vaginalis isolates of Spanish origin have been characterized using two molecular markers (microsatellites and single-copy genes), the presence of T. vaginalis virus (TVV) and Mycoplasma, and the resistance to reference drugs. On the basis of our molecular results, the isolates were classified into two groups, as proposed by other researchers. In relation to the biological determinations, the two isolates harboring Mycoplasma were sensible to the reference drugs and were included in the same genotypic group. Although the presence of TVV was more notable in one group than in the other, both groups had TTV+ and TVV- isolates. These findings indicate that genetic grouping does not strongly correlate with susceptibility to reference drugs or endobiont presence, suggesting the need for further research into alternative classification models.

Keywords: Mycoplasma; SNP; TVV; Trichomonas vaginalis; genotypes; microsatellites; resistance; single-copy gene.

Figure 1

Phylogenetic relationships of the GP63a sequences of Trichomonas vaginalis by Neighbor-Joining method. The number at the nodes represents the bootstrap support as computed from 1000 replicates. The tree is drawn to scale, with branch lengths measured in the number of base substitutions per site. Isolates grouped as GP63a-A or Type 1 are shown in red and GP63a-B or Type 2 in green. The sequences obtained in the present study are in bold.

Figure 2

Phylogenetic relationships of the PMS1 sequences of Trichomonas vaginalis by Neighbor-Joining method. The number at the nodes represents the bootstrap support as computed from 1000 replicates. The tree is drawn to scale, with branch lengths measured in the number of base substitutions per site. Isolates grouped as PMS1-A or Type 1 are shown in red and PMS1-B or Type 2 in green. The sequences obtained in the present study are in bold.

Figure 3

Determination of the presence of TVV endobiont in T. vaginalis isolates by indirect immunofluorescence using the anti-P270 monoclonal antibody C20A3: (a) Representative isolate positive for TVV, exhibiting green fluorescence on the cell surface; (b) Representative isolate negative for TVV, displaying red fluorescence from Evans blue staining.

Figure 4

Determination of the presence of Mycoplasma sp. endobiont in T. vaginalis isolates by the identification of bacterial DNA: (a,b) Negative isolates, showing a 270 pb band corresponding to the internal control (I.C.); (c) The two positive isolates to Mycoplasma (S760 and S351), showing a positive band at 500 pb.