Ethics statement
The present project is in compliance with the Helsinki Declaration (Ethical Principles for Medical Research Involving Human Subjects).
The study was conducted in accordance with the guidelines of the “Direction de la Recherche Clinique et de l’Innovation”, the research board of Bordeaux University hospital, Bordeaux, France. All patient data were anonymously reported, with no possibility of connecting the isolates and specimens to individual patients. Using the written “livret d’accueil” of the Bordeaux University Hospital, patients are explicitly informed at the admission to hospital that their samples could be used for research purposes and that they can oppose to this use. As specimens used in this study are part of routine patient management without any additional sampling, and since patients provided no objection for their samples to be used, the article L1211-2 of the French code of Public Health states that this study did not need to be examined by the ethical committee “Comité de Protection des Personnes” and that patient’s informed consent was not required.
Bacterial strains, culture and DNA preparation
The PG21 (ATCC 23114), M132 (ATCC 43521) and H34 (ATCC 15056) M. hominis reference strains and 207 French clinical isolates collected between 1987 and 2009 were used in this study (Additional file 1: Table S1). The 167 urogenital clinical isolates were collected at the Bordeaux University Hospital and obtained from i) specimens where M. hominis was present as a commensal, i.e. cervical samples with titres of M. hominis < 104 CCU /ml and male specimens, ii) cervical swabs from patients with titres of M. hominis ≥ 104 CCU /ml without association with BV, iii) cervical swabs from female patients with titres of M. hominis ≥ 104 CCU /ml and suffering from bacterial vaginosis, iv) vaginal swabs from pregnant women with threatened preterm delivery whatever the titre of M. hominis, v) specimens from women presenting upper genital tract infection whatever the titre of M. hominis, these specimens being normally sterile. Thirty-four isolates obtained from extragenital specimens and collected at hospitals from 10 different French cities were also tested. Finally, we genotyped six isolates obtained from two mother-neonate pairs.
Among these 210 isolates, concomitant and sequential isolates were obtained for one and seven patients, respectively.
Antibiotic susceptibility testing, realised when M. hominis was in a pathogenic situation, showed that 66 urogenital isolates were resistant to tetracyclines, seven extragenital isolates were resistant to ofloxacin, two urogenital isolates were resistant to both tetracyclines and ofloxacin and 91 isolates presented a wild-type profile.
The growth conditions used for the M. hominis isolates have been described previously [21]. The DNA was extracted using the MagNA Pure LC DNA isolation kit I (Roche, Meylan, France) according to the manufacturer’s instructions.
MLVA analysis
Tandem repeat (TR) sequences were identified in the M. hominis PG21 genome [20] using the Tandem Repeats Finder programme (http://tandem.bu.edu/trf/trf.html) [22]. Loci were chosen if they had >80% matches between the DNA sequences of the repeat units. A total of 130 TRs were selected and designated by the letters Mho followed by a number corresponding to the order in which the TR was detected. To screen for variability in the number of TRs, PCR primers targeting the regions flanking TR loci were designed and tested on a set of 12 M. hominis isolates, including the PG21, M132 and H34 references strains, three urogenital isolates, one from amniotic fluid, one isolate from peritoneal fluid, one from joint fluid, one from bronchial aspirate, one from abdominal wall abscess and one from a renal abscess, collected between 1995 to 2008 from different geographical areas (Additional file 1: Table S1). Each locus was amplified individually and analysed by conventional agarose gel electrophoresis.
To confirm that length polymorphisms were the result of repeat copy number variations, the PCR products were purified using the Wizard PCR Preps DNA Purification System (Promega, Charbonnières-les-Bains, France) and double-strand sequenced (Additional file 2: Figure S1). This approach showed that only seven loci were polymorphic with different allele sizes. After evaluation of a large collection of M. hominis isolates, two of these seven VNTRs were rejected due to a lack of adequate discrimination, and the five remaining VNTR loci were chosen for further assessment.
The five VNTR markers ultimately selected for use in MLVA were multiplexed in two solutions named T1 and T2. The markers Mho-50, Mho-52 and Mho-53 were amplified using the solution T1, and the markers Mho-114 and Mho-116 were amplified using the solution T2. The amplifications were performed with a Mastercycler ep Gradient S thermocycler (Eppendorf, Hamburg, Germany) in a final volume of 25 μl. The reaction mixtures contained 1X Qiagen PCR buffer with 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphate, 3 mM MgCl2, 0.625 U of Hot Start Taq DNA polymerase (Qiagen, Hilden, Germany), 0.125 μM of each primer and 1 μl of template DNA from clinical isolates. The forward primers were fluorescently labelled at the 5’ end using 4,4,7,2’,4’,5’,7’-hexachloro-6-carboxy-fluorescein (HEX), 6-carboxyfluorescein (FAM; Eurogentec, Angers, France) or NED (2’-chloro-5’-fluoro-7’,8’-fused phenyl-1,4-dichloro-6-carboxyfluorescein; Applied Biosystems, Life Technologies, Carlsbad, CA, USA), depending on the locus to be amplified (Additional file 3: Table S2). All of the solutions were run under the same cycling conditions: 95°C for 15 min followed by 25 cycles of 95°C for 1 min, 56°C for 1 min and 72°C for 1 min with a final extension at 72°C for 10 min. Prior to GeneScan analysis, 0.3 μl of GeneScan ROX 500 size standard (Applied Biosystems) was added to 1 μl of each PCR product. After heat denaturation for 5 min at 95°C, the fragments were separated using an ABI 3130 Genetic Analyzer (Applied Biosystems). The GeneScan data were subsequently analysed using GeneMapper software (version 3.7; Applied Biosystems) to perform sizing and to calculate the number of repeats in the PCR fragments. Each locus was identified according to colour fluorescence. An allele number string based on the number of repeats at each locus was assigned to each isolate.
Data analysis
The calculated numbers of repeats were imported into BioNumerics (version 6.1; Applied Maths). A minimum spanning tree (MST) was generated to visualise the relationships between the large number of isolates in a single compact image. The MST was created based on the categorical coefficient and a priority rule consisting of the highest number of single-locus variants. The polymorphism index of individual or combined VNTRs was calculated using the Hunter-Gaston discriminatory index (HGDI) [23].