In this study, we present an MLVA-based molecular typing system for the discrimination of M. hominis isolates. We used this method on a group of 210 temporally separated isolates from French patients and obtained from a variety of urogenital and extragenital clinical circumstances. This effort represents the most comprehensive M. hominis molecular typing study because, until now, other studies were realised only on urogenital isolates and few isolates were tested [7–10].
MLVA typing of M. hominis is important both individually and epidemiologically. At the level of an individual patient, this approach allows discrimination between relapse or persistence and new infections. In the first case, the MLVA type remains identical. In the case of a reinfection, the MLVA type is likely to be different. Our MLVA scheme was used to study the course of infection in seven patients. In six of these patients, sequential isolates belonged to a consistent MLVA type in each case studied, suggesting in a persistent or relapse infection. Interestingly, the two clinical isolates Mh-2377 and Mh-2477 harboured the unique MLVA type 33 whereas previous PFGE analysis showed different migrations patterns when evaluated according to the interpretation guidelines of Tenover et al., and the total genome sizes of the two strains, deduced from the addition of the generated fragment lengths, were nearly identical . These respiratory isolates were collected six months apart from a man with a chronic obstructive pulmonary disease who was treated several times with ciprofloxacin. As the M. hominis isolates were both resistant to fluoroquinolones, it would seem logical that the two isolates were identical, as shown by MLVA typing. The observed differences in PFGE patterns may be due to restriction sites located in variable regions or to recombination. Indeed, results from previous analysis indicated that a high levels of intragenic and intergenic recombination occurred in M. hominis, and these recombination levels are presumably important for the adaptation potential of this species [11, 14].
Analysis of our results suggests a new infection in a female patient, as the two sequential cervical isolates were of different MLVA types. A previous study investigated cervical isolates of M. hominis obtained before and after treatment by RAPD. In two of nine cases studied, the profile of amplification did not change, whereas in the rest of cases, RAPD patterns were different, suggesting that the patients were reinfected .
We also performed molecular investigations of M. hominis isolates from two mother-neonate pairs. In each case studied, an identical MLVA type was found, confirming mother-to-child transmission. Our results are in agreement with those of Jensen et al. who reported that M. hominis isolates obtained from the cervices of pregnant women and from the ears or pharynges of their new-born infants yielded the same genomic profile by PFGE . Similar results were obtained by Grattard et al., who showed that strains isolated within a mother-neonate pair exhibited an identical pattern by AP-PCR .
At the population level, MLVA typing assesses the genetic diversity of M. hominis strains. In this study, we described 40 MLVA types, revealing a genetic heterogeneity among this species. This finding is in agreement with the data obtained by studies using other molecular typing methods. Using RFLP, Busch et al. found a high heterogeneity among 20 isolates obtained from colonised women and women with various urogenital infections . The results from an AFLP analysis of five randomly chosen clinical isolates also provided further evidence of high-level intraspecies variability in this organism . Soroka et al. reported genetic heterogeneity of genomes of M. hominis isolates using RAPD, and their results were confirmed by PFGE . In comparison to the molecular typing methods that the other studies have used, MLVA is a reproducible and fast technique that does not require a sequencing step and can be standardised, facilitating large-scale molecular epidemiological investigations. The capillary electrophoresis on a genetic analyser enables high throughput analysis and allows easier interpretation of results (in contrast to agarose gel electrophoresis), particularly for VNTRs with a small number of repeat units.
In M. hominis, a high level of resistance to tetracyclines has been associated with the presence of the tet(M) determinant, the sole tetracycline resistance mechanism acquired by clinical isolates of human mycoplasmas . It has been reported that in Bordeaux, France, the percentage of M. hominis isolates resistant to tetracyclines increased significantly, from 2.8% to 18.75%, between 1999 and 2002 . In our study, the 68 urogenital M. hominis isolates resistant to tetracyclines were not related and clustered into 25 MLVA types, suggesting the absence of a link between tetracycline resistance and this typing method. Our results are in agreement with those of Mardassi et al., who recently showed that resistance rates to tetracyclines were 25% among Tunisian M. hominis isolates and that molecular typing based on the nucleotide sequences of P120’ gene fragments indicated that these isolates were not clonal .