Isolation, titration and cloning of M. hominisstrains
To determine the initial count of the mycoplasma species contained within the clinical samples, ten-fold dilutions of the original sample were performed and inoculated to SP-4 agar plates. Among the clinical specimens, 27 yielded a high number of viable mycoplasma varying from 105 to 106 C.F.U. (colony forming unit)/ml for M. hominis and only 5 M. genitalium and 12 U. urealyticum isolates were detected by culture with low number (≤ 104 C.F.U/ml) (Tab. 1). As mycoplasma cultures obtained from clinical specimens often represent a mixture of several species, especially with M. hominis and U. urealyticum, cloning was undertaken so that PCR amplification would involve a DNA template prepared from a single clone. Cloning is also crucial to obtain a real picture of the genetic heterogeneity within a single specie. For this purpose, single colonies from the highest (often the fifth or the sixth dilution) ten-fold dilutions of each specimen culture were picked, cultured into SP-4 broth medium, then diluted through 10 serial dilutions, and finally, 100 μl of each dilution was subcultured onto a SP-4 agar plate. This process was repeated thrice and allowed the appearance of typical Mycoplasma hominis colonies in these 27 high tittering clinical specimens. The colonies are clearly distinguishable from those of U. urealyticum which are characterized by their tiny size and dark colour.
PCR amplification and genetic diversity of the P120' surface exposed N-terminal coding sequence
All the 27 M. hominis cloned isolates yielded a PCR product of the expected molecular size (510 bp), suggesting that this gene sequence is highly conserved (Fig. 1). No amplification was observed using DNAs from other mycoplasma species namely, M. genitalium, M. fermentans, M. pneumoniae and U. urealyticum (Fig. 1, lanes 3–6), thus confirming the specificity of the PCR reaction. The nucleotide sequence encompassing the positions 145 to 565 (421 nucleotides) of the P120' gene sequence (amino acids 49 to 188) was obtained for each sample, by sequencing in both directions, two PCR products from independent amplifications. A total of 25 single nucleotide polymorphisms (SNPs) distributed through 23 polymorphic sites were observed (A162G, T177A, T192G, A212T, A212G, A215G, C234T, T297C, A300G, A300C, A379C, G394A, C396T, A399T, C405T, A409T, A417T, T441A, A482T, C483T, G500T, C509T, A552G, A563G, and T564A), yielding 13 haplotypes. Mutations at positions 234 (C to T) and 394 (G to T) were shared by the 27 isolates, whereas the SNPs at positions 297, 300, 379, 396, 405, 500, and 552 were found in more than 20 isolates.
Among the 23 polymorphic sites, thirteen (56.5%) are non synonymous as they cause an amino acid change (D59E, N64K, E71V, E71G, N72S, K100N, K127Q, V132I, I137F, E139D, D161V, R167I, A170V, and N188R) (Fig. 2). Of these, mutations K127Q, V132I, A170V, and N188K were shared by most of the isolates. Overall, the mutations were mainly confined within three regions, 59–72, 127–139, and 161–188, each containing four amino acid changes. We could distinguish 10 haplotypes among the deduced amino acid sequences.
Genetic relatedness of M. hominisisolates and evolution of the N-terminal region of P120'
The unrooted neighbour-joining-based phylogenetic derived tree (Fig. 3), using the deduced amino acid sequences of the 27 M. hominis isolates, showed a major branch containing a group of 17 closely-related isolates and 6 variant emerging strains. This major branch, which is supported by bootstrap, is clearly distinct from that of the reference M. hominis strain PG21. The remaining isolates are less phylogentically close to the majority of the pool; Mh13 and Mh8 belonging to a distinct evolutionary tract.
The acquisition rates for sSNP (Ks) and nsSNP (Ka) were calculated in order to evaluate the evolutionary trend of this surface exposed part of the P120' protein. We found that the overall mean value of 0.046 for Ks (0.011–0.47) is significantly different (P < 0.0001) from the 0.012 mean value of Ka (0.003–0.18). The ratio Ka/Ks (0.26) is thus <1, indicative of a negative selection. This finding is in accordance with a previous study involving house-keeping genes [24].