Genetic variability of the P120' surface protein gene of Mycoplasma hominis isolates recovered from Tunisian patients with uro-genital and infertility disorders

Five reference strains of human mycoplasmas, three ureaplasma isolates, and 27 mycoplasma clinical isolates were used: Reference strains were purchased from the American Type Culture Collection (ATCC): M. hominis PG21 (ATCC 23114), M. fermentans (ATCC 19989), M. genitalium (ATCC 33530), M. pneumoniae (ATCC 15531), and Ureaplasma urealyticum, isolate UuipT3, recovered in our laboratory. Reference and isolated mycoplasma strains were cultivated in SP-4 medium [16] supplemented with 5% of CMRL 1066 (Sigma), 2000 units/ml of penicillin G, 500 units/ml of polymyxin B, 2.5 μg/ml amphotericin B, 10% of fresh yeast extract (Amersham Biosciences), 15% of horse serum (Gibco BRL), and 0.5% phenol red. The medium was further supplemented with 0.5% of glucose, 0.5% of arginine, or 0.5% of urea depending on the nutritional needs of the species being cultivated. Broth culture was done at 37°C and mycoplasma growth confirmation was performed onto SP-4 agar plates maintained at 37°C with 5% CO₂ and regularly observed microscopically for the appearance of mycoplasma colonies.

Cervical swabs from 34 female patients suffering from sexually transmissible disease, 23 vaginal specimens from women presenting a number of complications associated to pregnancy, including chorioamnionitis and preterm birth, 29 urethral specimens obtained from men with urethritis, and 6 semen samples from clinical cases associated with infertility. All these specimens were tested for the presence of genital mycoplasmas (M. hominis, M. genitalium, M. fermentans, and Ureaplasma urealyticum) by broth and solid culture, then by PCR. The collected specimens were inoculated onto 1.8 ml SP-4 broth medium and then transferred to the laboratory at 4°C within 24 h. Semen samples were also placed in a tube with 1.8 ml SP-4 medium for cultivation attempts. The specimens were processed on arrival or after storage at -80°C. After filtration through 0.45 μm single use syringe filter, 200 μl of the specimen was inoculated into each of SP-4 broth with 0.5% of glucose and 0.5% of arginine for the isolation and identification of mycoplasmas and SP-4U broth with 0.5% of urea for Ureaplasma urealyticum isolation and identification. In parallel, 100 μl of the filtrated specimen was inoculated onto each of SP-4 and SP-4U agar plate for the monitoring of mycoplasma or ureaplasma colonies appearance. All broth cultures were incubated at 37°C and examined daily for turbidity and pH change. The broth cultures were subcultured onto agar plates once their pH changes. The plates were incubated at 37°C in a 5% carbon dioxide environment and examined every two days for the appearance of mycoplasmas or ureaplasma colonies. To confirm the identification of Mycoplasma spp or ureaplasma, PCR assays have been used on positive broth cultures. For mycoplasma titration, dilution culture and subculture were achieved as described elsewhere [17].

This study has been conducted in close conformity with ethical aspects, which were established by the ethical committee of the Tunisian Ministry of Health. The samples were collected in the context of the routine diagnostic activity of the laboratory of Mycoplasma and with the consent of the patients. All the patient files are confidential and none could be identified.

Culture (8 ml) of the cloned mycoplasma strains and clinical samples was centrifuged at 17 000 rpm for 30 min, washed in PBS (0.1 M-NaCl, 2.5 mM-KCl, 10 mM-Na₂HPO₄, 1.5 mM-KH₂PO₄, pH 7.4), and then suspended in 200 μl of PBS. This concentrated culture was processed for PCR as described elsewhere [18]. Briefly, SDS to a final concentration of 1% was added to the DNA and incubated at 37°C for half an hour in the presence of RNase A (5 μg/ml). A volume of 25 μl was then used for PCR amplification.

Two pairs of primers were used in this study. The pair Mhp120'F (5'-GAGGAATTTCAACTGGTGTCC-3') and Mhp120'R (5'-CTGTTGTAATAGCATTTAAG-3') was specific to the M. hominis p120' gene. PCR was performed on 25 μl of treated sample in a total volume of 50 μl containing, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 250 μM of each dNTP (Amersham Biosciences), 50 pmol of each primers, and 1.25 U of Taq DNA polymerase (Amersham Biosciences). The PCR mixtures were subjected to 30 cycles consisting of 1 min at 95°C, 2 min at 50°C, and 1 min at 72°C in a Perkin-Elmer GeneAmp PCR System 9700 thermocycler. After the last cycle, a final extension temperature of 72°C was maintained for 10 min. To determine the specificity of M. hominis primers, the PCR reaction was performed with DNAs from other mycoplasma species, M. fermentans, M. genitalium, M. pneumoniae, which occasionally has been isolated from urogenital tract, and Ureaplasma urealyticum, strain UuipT3. The PCR products were analysed on 2% agarose gels. The PCR experiments were repeated at least twice for results confirmation.

The PCR products were separated by electrophoresis in a 1.5% low-melting-point agarose gel, excised from the gel, and then purified using the GFX PCR DNA and Gel Band Purification system (Amersham Biosciences). Determination of the nucleotide sequence was performed with the Prism Ready Reaction Dye Deoxy Terminator Cycle sequencing Kit on an ABI PRISM 377 DNA sequencer (Applied Biosystems). Each sample was sequenced from two independent PCR amplification reactions.

The sequence data (GenBank accession numbers; pending) were aligned using ClustalW [19] and edited with the software programs BioEdit [20]. DNASP [21] was used to calculate haplotypes and to test for adaptive selection, by determining the nucleotide substitution changes and the ratio of synonymous (Ks) and nonsynonymous (Ka) substitutions per site. For this purpose, we used the analysis developed by Nei-Gojobori [22] as implemented in the DNASP package. A dendrogram illustrating the genetic relatedness of the recovered M. hominis isolates was constructed using MEGA version 3.0. [23]. The dendrogram was obtained using the neighbor-joining method with 1000 bootstrapping replicates.

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.

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.

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].