Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity

Sampling.Biological samples were collected from the oral cavity of 23 healthy women and 21 men between 25 and 38 years of age. Approval (agreement no. 2016-010) was obtained from the local ethics committee of the IHU-Mediterranée Infection (Marseille, France). Donors were healthy volunteers who did not have symptoms of gingivitis and/or periodontitis. Oral specimens were collected using a sterile toothbrush in the morning before brushing. After sampling, each toothbrush was placed in a tube containing 10 ml of oral Treponema enrichment broth (OTEB) (18) supplemented with ascorbic acid (1 g/liter), uric acid (0.1 g/liter), and glutathione (0.1 g/liter), to protect anaerobic bacteria during transport (19). The tubes were then closed and placed in a plastic bag containing an anaerobic generator (Thermo Scientific, Dardilly, France), and samples were analyzed in the laboratory immediately after reception (approximately 1 h after sampling).

Sample preparation for electron microscopy.Smear samples were directly swabbed from the oral cavity onto microscope slides. We then added one drop of 2.5% glutaraldehyde fixative solution and proceeded to imaging without any additional staining. Regarding the culture of Treponema, the bacteria were later directly collected from culture-medium-adapted tubes and immersed in a 2.5% glutaraldehyde fixative solution. A drop of the suspension was then directly deposited onto a poly-l-lysine-coated microscope slide for 5 min. Subsequently, images were obtained directly or with the addition of 1% phosphotungstic acid (PTA) in aqueous solution (pH 2) for 2 min for improved quality. The slide was then gently washed in water, air dried, and examined by tabletop scanning electron microscopy (SEM). This procedure was performed to increase SEM image contrast, if necessary.

Detection, observation, and image acquisition by SEM.We used a tabletop SEM system (Hitachi TM4000, approximately 33 cm wide by 60 cm tall) to evaluate the bacterial structures. The SEM has a capability of observing specimens under low vacuum pressure (10° Pa to 10¹ Pa), to reduce charge on the specimen’s surface from irradiated electrons. The evacuation time after loading the specimen into the SEM chamber is a few minutes. This is much faster than conventional SEMs with high vacuum conditions directly installed on the floor, as the chamber of the tabletop SEM is smaller than that of the conventional type. The optimized observation conditions of acceleration voltage and electron beam current make it possible to operate the SEM easily and quickly. Indeed, it only takes a few minutes to find and observe the regions of interest, while conventional SEMs with high-performance specifications require fine but complicated and specific adjustments of focus and stigma. SEM images were obtained in the magnification range of ×1,000 to ×10,000. Backscattered electrons (BSEs) coming from the specimen’s surface were detected by a BSE detector at an accelerating voltage of 15 kV. The vacuum around the sample was approximately 30 Pa.

Optimization of Treponema culture medium.To optimize the new culture medium used here, we compared the compositions of the four most used media for cultivating Treponema organisms, namely, OTEB (18, 20), OMIZ-Pat medium (21), new oral spirochaetae (NOS) medium (22), and spirochaetae medium (23), by using a Venn diagram (http://bioinformatics.psb.ugent.be/webtools/Venn). This analysis identified the common and essential nutrients for the growth of Treponema organisms and the specific elements for each medium. Then, all of the common and specific components were retained to prepare our culture medium. The T-Raoult culture medium is composed of 5 g proteose peptone, 5 g brain heart infusion, 5 g yeast extract, 2 g Casamino Acids, 2 g K₂HPO₄, 5 g NaCl, 0.1 g MgSO₄, 2 g NaHCO₃, 0.5 g l-cysteine, 0.5 g Na₂S, 0.5 g sodium pyruvate, 0.5 g sodium thioglycolate, 1.6 g acetic acid, 0.6 g propionic acid, 0.4 g n-butyric acid, 0.1 g n-valeric acid, 0.1 g isovaleric acid, 0.1 g isobutyric acid, 0.1 g 2-methylbutyric acid, 0.4 g glucose, 0.4 g fructose, 0.4 g fucose, 0.4 g sucrose, 0.4 g maltose, 0.4 g ribose, 0.4 g xylose, 0.4 g mannitol, 0.4 g mannose, 0.4 g arabinose, 0.4 g fucose, 0.4 g rhamnose, 0.4 g trehalose, 0.4 g pectin, 0.4 g soluble starch, 1 g ascorbic acid; 0.1 g uric acid, and 0.1 g glutathione. All compounds were prepared in 900 ml of distilled water and filtered using a 0.2-μm microfilter (Thermo Scientific, Waltham, MA, USA). We then added 0.05 mg vitamin B₁₂, 0.05 mg d-(+)-biotin, 0.05 mg folic acid, 0.05 mg folinic acid, 0.05 mg nicotinamide, 0.1 mg nicotinic acid, 0.01 mg riboflavin, 0.1 mg vitamin K₁, and 0.5 mg thiamine pyrophosphate. This medium was supplemented with 100 ml of calf serum inactivated by incubation at 56°C for 60 min and filtered through 0.1-μm-pore filters (Corning bottle-top vacuum filter system; Sigma, France). The following antibiotics were also added, to inhibit the growth of other bacteria and to eliminate contaminants from the oral flora: rifampin (2 mg/liter), nalidixic acid (500 mg/liter), and polymyxin B (5 mg/liter).

Isolation by passive filtration.The enrichment culture in liquid medium was carried out anaerobically in an anaerobic chamber (Don Whitley Scientific Ltd., Bingley, UK), using a new culture device based on a passive filtration principle and consisting of two chambers separated by a 0.22-μm microfilter (Dominique Dutscher, Brumath, France). The bottom compartment of each inoculated filtration unit was inspected daily with electron microscopy (Hitachi TM4000), at magnifications of ×1,000 to ×10,000, to highlight the presence of Treponema species.

Culture and purification of bacterial colonies.After the primary isolation of the Treponema species in liquid medium, agar cultures were carried out using the same liquid medium supplemented with 7 g/liter agar (Fisher Scientific, Illkirch, France). Endpoint dilutions up to 10⁻¹⁰ for each positive liquid culture were confirmed by electron microscopy and carried out for all dilutions (900 μl), which were finally inoculated into a tube containing 25 ml of solid medium that had been previously prepared and maintained at 56°C. The mixture was gently homogenized and poured directly into Petri dishes. After solidification, inoculated Petri dishes were incubated in the anaerobic chamber at 37°C for 5 days (Fig. 1). Single colonies were recovered from the agar using a sterile Pasteur pipette (Biosigma, Brumath, France), and the presence of Treponema was determined by electron microscopy. Each colony was inoculated onto streak agar plates and transferred into Hungate tubes (20) containing liquid medium, in order to obtain pure cultures of Treponema species. This procedure was repeated regularly every 4 days.

• Open in new tab
• Download powerpoint

FIG 1

Different culture steps and identification of Treponema strains. (a) Sampling. (b) Direct observation and detection by SEM (Hitachi TM4000). (c) SEM image, showing the presence of Treponema in oral samples. (d) Procedure for primary isolation by passive filtration in liquid medium, with observation of the culture using SEM (Hitachi TM4000). (e) SEM image, showing the presence of Treponema in the culture suspension at a higher proliferation rate. (f) Method for solid culture (0.7% agar). (g) Culture and purification of single colonies. (h) Control of the purity of the culture. (i) Conservation of the strain at –80°C. (j) Genome sequencing and molecular identification. (k) MS MALDI-TOF analysis and identification of the strain of Treponema.

Determination of incubation times.In order to collect a fresh culture of Treponema and to limit bacterial contamination by filtration, a positive oral sample for Treponema was cultured on five filtration units at the same time, using the previously prepared liquid T-Raoult medium. A culture unit was closed every 24 h and the presence of the Treponema was checked by optical and electron microscopy, as indicated previously. This experiment was performed in triplicate.

Treponema organisms.Treponema brennaborense DSM12168, Treponema maltophilum DSM27366, Treponema parvum DSM16260, Treponema pedis DSM18691, Treponema zuelzerae DSM1903, Treponema succinifaciens DSM2489, Treponema stenostreptum DSM2028, Treponema saccharophilum DSM2985, Treponema ruminis DSM103462, Treponema rectale DSM103679, Treponema caldarium DSM7334, Treponema bryantii DSM1788, and Brachyspira hyodysenteriae DSM105803 were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ) (Braunschweig, Germany). The strains were cultured first in the recommended DSMZ liquid medium and then, using the new culture system, in the T-Raoult culture medium reported here.

Molecular and MALDI-TOF MS identification.(i) Genome assembly and annotation. Total paired-end reads, filtered according to read qualities, were assembled using SPAdes 3.10.1 software (24). To run SPAdes, we carefully used the pipeline options to reduce the numbers of mismatches and short indels; all other parameters were set with default options. Open reading frame (ORF) prediction and gene annotation were carried out using Prodigal software in the Prokka annotation suite (http://www.vicbioinformatics.com/software.prokka.shtml), with the Clusters of Orthologous Groups (COG) database as a reference (25). Barrnap (http://www.vicbioinformatics.com/software.barrnap.shtml) and tRNAscan-SE (26) were used to identify rRNA and tRNAs. For genome visualization, the chromosome topology was drawn using DNAPlotter (27).

(ii) Pan-genome analysis. We determined the pan-genome composition of five T. pectinovorum strains, namely, ATCC 700769, Marseille1-CSURP6641, Marseille2-CSURP7641, Marseille3-CSURP7641, and Marseille4-CSURP7639. These compositions were determined using Roary (28), with default parameters.

(iii) Digital DNA-DNA hybridization. To assess the genomic similarity among the genomes studied, we determined the digital DNA-DNA hybridization (dDDH), which exhibits a high correlation with DNA-DNA hybridization (DDH) (29, 30), when dDDH is >70% (i.e., same species) and when dDDH is >79% (i.e., same subspecies) (30).

(iv) MALDI-TOF MS analysis. For direct identification from the liquid medium, 2 ml of the liquid culture was centrifuged at 13,000 rpm for 5 min. The supernatant was discarded, and the bacterial pellets were recovered. Twelve spots were then made for each bacterial pellet and coated with 1 μl of matrix solution (31) before being analyzed by MALDI-TOF MS (Bruker Daltonics, Bremen, Germany). After analysis of the protein profile of each Treponema isolate, the spectra generated by MALDI-TOF MS without initial identification were recovered to control their quality. After validation of the spectra by the Bruker software, MALDI Biotyper 3.0 software was used to build dendrograms, which allowed comparison of the different isolates (31). Finally, one isolate from each sample was selected for genome sequencing. As a result of this procedure, only five different isolates had their genomes sequenced and analyzed to establish a 16S rRNA-based identification. After 16S rRNA identification, the spectra were added to our MALDI-TOF MS database.

Accession number(s).The draft genome sequences of T. pectinovorum ATCC 700769, T. pectinovorum Marseille1-CSURP6641, T. pectinovorum Marseille2-CSURP6642, T. pectinovorum Marseille3-CSURP764, T. pectinovorum Marseille4-CSURP7639, and T. denticola Marseille5-CSURP7640 were deposited at EMBL-EBI under accession numbers UPSF01000000, UOUI01000000, UFQJ01000000, UFQO01000000, UFQN01000000, and UFQP01000000, respectively.