Molecular epidemiology of metronidazole resistance in a population of Trichomonas vaginalis clinical isolates

Abstract

Trichomonas vaginalis, the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women, where it may be asymptomatic or cause severe vaginitis and cervicitis. Despite its high prevalence, the genetic variability and drug resistance characteristics of this organism are poorly understood. To address these issues, genetic analyses were performed on 109 clinical isolates using three approaches. First, two internal transcribed spacer (ITS) regions flanking the 5.8S subunit of the ribosomal DNA gene were sequenced. The only variation was a point mutation at nucleotide position 66 of the ITS1 region found in 16 isolates (14.7%). Second, the presence of a 5.5-kb double-stranded RNA T. vaginalis virus (TVV) was assessed. TVV was detected in 55 isolates (50%). Finally, a phylogenetic analysis was performed based on random amplified polymorphic DNA data. The resulting phylogeny indicated at least two distinct lineages that correlate with the presence of TVV. A band-sharing index indicating relatedness was created for different groups of isolates. It demonstrated that isolates harboring the virus are significantly more closely related to each other than to the rest of the population, and it indicated a high level of relatedness among isolates with in vitro metronidazole resistance. This finding is consistent with the hypothesis that drug resistance to T. vaginalis resulted from a single or very few mutational events. Permutation tests and nonparametric analyses showed associations between metronidazole resistance and phylogeny, the ITS mutation, and TVV presence. These results suggest the existence of genetic markers with clinical implications for T. vaginalis infections.

FIG. 1

Schematic representation of the rDNA gene of T. vaginalis. The rDNA gene of eukaryotic organisms is arranged in tandem repeats. The ITS regions are transcribed with the gene and then are removed before the ribosome is assembled. In the rDNA gene of T. vaginalis, ITS1 begins at nucleotide position 1578 and ends at nucleotide position 1658. A point mutation at the 66th position of the ITS1 region (nucleotide position 1644 of the rDNA gene), in which a cytosine is replaced by a thymidine, occurs in less than 15% of the isolates.

FIG. 2

Representative agarose gel with OPD3 RAPD products. At least two separate PCRs were performed for every isolate using each primer. The gel shows the banding pattern of the PCR using OPD3 for 18 isolates. The lane on the far left was loaded with a 1-kb DNA ladder (Promega).

FIG. 3

Average MLC for isolates with different characteristics. (A) The average MLCs of isolates grouped based on the presence or absence of TVV. (B) The average MLCs of isolates grouped based on the presence or absence of the rDNA ITS C66T mutation. Isolates with MLC values of >400 μg/ml were treated as 400 μg/ml.

FIG. 4

Dendrogram for MSA isolates based on RAPD data. The tree was built by the neighbor-joining method. The distance matrix was calculated with Pearson's phi coefficient based on RAPD data from three primers (29 bands). Isolates negative for TVV (italicized) all aggregated in the same (upper) branch, and TVV-positive isolates grouped together in a separate branch. All isolates with the rDNA ITS C66T mutation (underlined) also sorted into the main upper branch. Branch lengths are drawn to scale based on genetic distances between isolates.

FIG. 5

Dendrogram for ATL isolates based on RAPD data. The tree was built by the neighbor-joining method. The distance matrix was calculated with Pearson's phi coefficient based on RAPD data from three primers (29 bands). All isolates negative for TVV (italicized), except ATL170, aggregated in one branch. All TVV-positive isolates grouped together in the main lower branch. Isolates with the rDNA ITS C66T mutation (underlined) were again associated with the TVV-negative isolates in the main upper branch. Branch lengths are drawn to scale based on genetic distances between isolates.