Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

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(1)Identification and Subtyping of Clinically Relevant Human and Ruminant Mycoplasmas by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry S. Pereyre,a,b,c F. Tardy,d,e H. Renaudin,a,b,c E. Cauvin,f L. Del Prá Netto Machado,a,g A. Tricot,e,d F. Benoit,f M. Treilles,f* C. Bébéara,b,c Université de Bordeaux, USC EA371 Infections Humaines à Mycoplasmes et Chlamydiae, Bordeaux, Francea; INRA, USC Infections Humaines à Mycoplasmes et Chlamydiae, Bordeaux, Franceb; Centre Hospitalier Universitaire de Bordeaux, Laboratoire de Bactériologie, Bordeaux, Francec; Anses, Laboratoire de Lyon, UMR Mycoplasmoses des Ruminants, Lyon, Franced; Université de Lyon, VetAgro Sup, UMR Mycoplasmoses des Ruminants, Marcy L’Etoile, Francee; Laboratoire Départemental de la Manche, Service Santé Animale, Saint-Lô, Francef; Programa de Pós Graduação em Farmácia, Universidade Federal de Santa Catarina, Florianópolis, Brazilg. I. n recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has emerged as a new technology for species identification. MALDI-TOF MS is a rapid, inexpensive, and accurate approach for species-level identification of Gram-negative and Gram-positive bacteria and yeasts (1). It has been recently optimized for routine use in clinical microbiology laboratories (2, 3). This method has shown to be sufficiently robust to be applicable with fastidious bacteria, such as anaerobic bacteria, Legionella, Bartonella, or mycobacteria (4). Mycoplasmas are wall-less bacteria with minimal genomes but are nonetheless important pathogens in both human and veterinary medicine (5). The family Mycoplasmataceae of the class Mollicutes regroups two major genera, Mycoplasma and Ureaplasma (6), which will be indicated here by their trivial name, mycoplasmas. The identification of human and ruminant mycoplasmas is challenging, and phenotypic methods cannot always achieve identification to the species level. Furthermore, in contrast to more classical bacteria, mycoplasmas express few biochemical traits that are useful for diagnosis. Nonetheless, several biochemical procedures have been standardized, such as the hydrolysis of urea and arginine, fermentation of glucose (7), and phosphatase activity (8), yet these may be time-consuming and not always discriminating. For example, it is not possible to distinguish Ureaplasma parvum from Ureaplasma urealyticum in culture because they both hydrolyze urea; it is likewise not possible to distinguish Mycoplasma pneumoniae from Mycoplasma amphoriforme in culture because they both grow in Hayflick modified broth medium sup-. 3314. jcm.asm.org. Journal of Clinical Microbiology. plemented with glucose (HG medium). Consequently, to identify these species easily, important efforts have been made over the past decades to develop diagnostic tools that rely on the antigenic (9, 10) and molecular identification of isolates, mainly based on PCR assays (7, 11–14). Nevertheless, the identification of certain mycoplasmas, such as subspecies belonging to the Mycoplasma mycoides cluster, comprising several important ruminant pathogens that are closely related both antigenically and genetically, has remained problematic (15, 16). Although genes encoding 16S rRNA have long been the usual targets for universal PCRs followed by sequence or amplicon analysis (17, 18), species-specific PCRs targeting 16S rRNA or other housekeeping genes are often favored because the techniques are more rapid and do not require a specialized laboratory (for reviews, see references 12 and 7). However, with ⬎100 mycoplasmas that are currently recognized. Received 17 June 2013 Returned for modification 7 July 2013 Accepted 25 July 2013 Published ahead of print 31 July 2013 Address correspondence to S. Pereyre, sabine.pereyre@u-bordeaux2.fr, or F. Tardy, florence.tardy@anses.fr. * Present address: M. Treilles, Laboratoire d’Analyses de Sèvres Atlantique, Niort, France. S.P. and F.T. contributed equally to this work. Copyright © 2013, American Society for Microbiology. All Rights Reserved. doi:10.1128/JCM.01573-13. p. 3314 –3323. October 2013 Volume 51 Number 10. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) recently emerged as a technology for the identification of bacteria. In this study, we aimed to evaluate its applicability to human and ruminant mycoplasmal identification, which can be demanding and time-consuming when using phenotypic or molecular methods. In addition, MALDI-TOF MS was tested as a subtyping tool for certain species. A total of 29 main spectra (MSP) from 10 human and 13 ruminant mycoplasma (sub)species were included in a mycoplasma MSP database to complete the Bruker MALDI Biotyper database. After broth culture and protein extraction, MALDI-TOF MS was applied for the identification of 119 human and 143 ruminant clinical isolates that were previously identified by antigenic or molecular methods and for subcultures of 73 ruminant clinical specimens that potentially contained several mycoplasma species. MALDI-TOF MS resulted in accurate (sub)species-level identification with a score of >1.700 for 96% (251/262) of the isolates. The phylogenetically closest (sub)species were unequivocally distinguished. Although mixtures of the strains were reliably detected up to a certain cellular ratio, only the predominant species was identified from the cultures of polymicrobial clinical specimens. For typing purposes, MALDI-TOF MS proved to cluster Mycoplasma bovis and Mycoplasma agalactiae isolates by their year of isolation and genome profiles, respectively, and Mycoplasma pneumoniae isolates by their adhesin P1 type. In conclusion, MALDI-TOF MS is a rapid, reliable, and cost-effective method for the routine identification of high-density growing mycoplasmal species and shows promising prospects for its capacity for strain typing..

(2) Identification of Mycoplasmas by MALDI-TOF MS. TABLE 1 Reference strains used to generate the mycoplasma MSP database with their respective phylogenetic group, usual host, and culture medium Main host. Strain. Source or ATCC/NCTC no.. Growth mediumc. Hominis Mycoplasma agalactiae M. agalactiae Mycoplasma alkalescens Mycoplasma arginini Mycoplasma bovigenitalium Mycoplasma bovirhinis Mycoplasma bovis M. bovis Mycoplasma canadense Mycoplasma canis Mycoplasma fermentans Mycoplasma hominis M. hominis M. hominis Mycoplasma orale Mycoplasma ovipneumoniae Mycoplasma salivarium. Ovine/caprine Ovine/caprine Bovine Several animal hosts Bovine Bovine Bovine Bovine Bovine Canine/bovine Human Human Human Human Human Ovine/caprine Human. PG2 5632 PG51 G230 PG11 PG43 PG45 1067 275C PG14 PG18 PG21 H34 M132 CH 19299 14811 PG20. NCTC 10123 Nouvel et al. (30) ATCC 29103 ATCC 23838 ATCC 19852 ATCC 27748 ATCC 25523 Hermeyer et al. (32) ATCC 29418 ATCC 19525 ATCC 19989 ATCC 23114 ATCC 15056 ATCC 43521 ATCC 23714 Strain being sequencedb ATCC 23064. Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO HG medium HA medium HA medium HA medium HA medium Modified PPLO HA medium. Spiroplasma Mycoplasma capricolum subsp. capricolum Mycoplasma mycoides subsp. capri Mycoplasma mycoides subsp. capri M. putrefaciens Mycoplasma yeatsii. Caprine Caprine Caprine Caprine Caprine. California kid GM12 PG3 KS1 GIH. ATCC 27343 ATCC 35297 NCTC 10137 ATCC 15718 ATCC 51346. Modified PPLO Modified PPLO Modified PPLO Modified PPLO Modified PPLO. Pneumoniae Mycoplasma amphoriforme Mycoplasma genitalium Mycoplasma penetrans Mycoplasma pneumoniae M. pneumoniae Ureaplasma parvum Ureaplasma urealyticum. Human Human Human Human Human Human Human. MA-3663 G37 GTU-54 M129 FH 27 T960. Pereyre et al. (13) ATCC 33530 ATCC 55252 ATCC 29342 ATCC 15531 ATCC 27815 ATCC 27618. HG medium HG medium HG medium HG medium HG medium Shepard medium Shepard medium. Other information. Adhesin P1 type 1 Adhesin P1 type 2 Serovar 3 Serovar 8. a. Inferred from 16S rRNA gene sequences as defined in reference 25. Bioproject accession no. PRJNA164759. c HA, Hayflick modified broth supplemented with arginine; HG, Hayflick modified broth supplemented with glucose. The compositions of the media are described in references 7 and 9. Culture conditions were 37°C under a normal atmosphere for human species or 5% CO2 for ruminant species to the mid-log phase. b. as pathogens in humans and animals, individual PCR-based systems that were developed as species-specific tests might be difficult to handle in a laboratory; thus, the quest for a single generic test remains pressing. MALDI-TOF MS might be an interesting universal alternative for mycoplasma (sub)species-level identification, though its use has been hindered thus far by a lack of databases of mycoplasmal MALDI-TOF reference spectra. The technique, per se, has been assessed only once for the identification of Mycoplasma pulmonis, a rodent mycoplasma species, using a limited database of three MALDI-TOF spectra from three rodent pathogen species (19). Indeed, no ruminant or human mycoplasma identification using MALDI-TOF MS has been reported to date, even though these bacteria represent important pathogens for these hosts (5). The aim of this study was to determine whether MALDI-TOF MS is a useful tool for the identification of ruminant and human mycoplasmas at the species or subspecies level and as a subtyping method for certain species. A total of 29 reference peak lists, or main spectra (MSPs), from 10 human and 13 ruminant mycoplasma (sub)species were first generated to construct a myco-. October 2013 Volume 51 Number 10. plasma spectral database and complete the database of the Bruker MALDI Biotyper platform. MALDI-TOF MS was then applied for identification purposes to 119 human and 143 ruminant clinical isolates that were previously identified to the species level using phenotypic, antigenic, or molecular techniques and to 73 subcultures of clinical specimens known to potentially contain more than one species of ruminant mycoplasmas. All analyses were optimized to be compatible with the routine workflow of our laboratories. The accuracy of subtyping was assessed using clinical strains of M. pneumoniae, Mycoplasma bovis, and Mycoplasma agalactiae. MATERIALS AND METHODS Reference strains, clinical isolates, and specimens. The reference strains used to generate MSPs are listed in Table 1. All strains were grown at 37°C in diverse media, and several strains were used as the reference strains. For comparison purposes, a maximum likelihood phylogenetic tree of the 16S rRNA sequences of these strains or species was computed with the MEGA5 software (http://www.megasoftware.net/). All the 16S rRNA sequences were downloaded from the Ribosomal Database project. jcm.asm.org 3315. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. Phylogenetic groupa and (sub)species.

(3) Pereyre et al.. 3316. jcm.asm.org. mycoplasma pellets were washed once for the human mycoplasmas and twice for the ruminant mycoplasmas in 1⫻ phosphate-buffered saline (PBS) prior to being suspended in 300 ␮l of PBS or water, and 900 ␮l of absolute ethanol was added to precipitate the proteins. After centrifugation at 20,000 ⫻ g for 2 min, the supernatant was removed and the pellet was dissolved in an equal volume (20 ␮l or 50 ␮l) of 70% formic acid and acetonitrile before centrifugation for 2 min at 20,000 ⫻ g. One microliter of the supernatant was spotted onto a MALDI MSP 96 target polished steel plate (Bruker Daltonics, Bremen, Germany). After air-drying at room temperature, each sample was overlaid with 1 ␮l of matrix solution composed of 10 mg/ml ␣-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 2.5% trifluoroacetic acid and air dried prior to the MALDI-TOF MS measurement. MALDI-TOF mass spectrometry. Peptide mass fingerprint product ion spectra were generated using a Microflex LT Biotyper operating system (Bruker Daltonics, Bremen, Germany). The data were analyzed in the automatic mode using the Bruker Biotyper 3.0 software and both taxonomy libraries, i.e., that from Bruker and that generated in the present study. A bacterial test standard (BTS) (part no. 255343; Bruker Daltonics, Inc.) was used in each run as a calibrator and for quality control. Mycoplasma MSP database construction and isolate identification by MALDI-TOF MS. Reference peak lists were assigned to different reference strains (Table 1) to construct the mycoplasma MSP database, which was used in addition to the Bruker MALDI Biotyper MSP database 3.1.2.0 (January 2011) for species-level identification of the clinical samples. The MSPs were created according to the manufacturer’s recommendations by using the automated MSP creation functionality of the MALDI Biotyper software package (version 3.0). For each reference strain, 20 individual mass spectrum measurements from 20 different spots of protein extracts from human mycoplasma species or three independent mass spectrum measurements of eight different spots of protein extracts from ruminant mycoplasma were acquired. The quality of individual raw spectrum measurements was carefully checked (for absence of intrusive peaks, absence of flat-line spectra, low matrix background signal) and a minimum of 18 spectra of high quality were selected for MSP creation. After smoothing, baseline correction, and peak picking, the resulting peak lists were used by the program to calculate and store an MSP containing the information about mean peak masses, mean peak intensities, and peak frequencies. For the identification of clinical isolates, the protein extracts were spotted once, twice, or 10 times, according to the species to be analyzed, and were classified by matching MSPs. The degree of spectral concordance was expressed as a logarithmic identification score ranging from 0 to 3 and was interpreted according to the manufacturer’s instructions, with a modification of the score that was acceptable for probable specieslevel identification, which was lowered from ⱖ2.000 to ⱖ1.700. The dendrograms were generated using the Biotyper 3.0 software. They were used to infer the relationships of mycoplasma species or strains, the closeness of which is reflected by an arbitrary distance level calculated by the software. This distance level is a relative value and thus cannot be compared between dendrograms.. RESULTS. Human and ruminant mycoplasma MSP database construction. To create the human and ruminant mycoplasma MSP database, 29 strains (listed in Table 1) were cultured in an appropriate medium prior to protein extraction, MALDI-TOF MS, and MSP generation. These strains represent 10 human and 13 pathogenic or commensal mycoplasma (sub)species belonging to different phylogenetic groups based on 16S rRNA sequences, as illustrated in Fig. 1A. To control the quality of this database, the MSPs generated were further used to perform a hierarchical clustering of strains using the MSP dendrogram tool of the MALDI Biotyper software. The resulting score-oriented dendrogram (Fig. 1B). Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. (http://rdp.cme.msu.edu/index.jsp) or from the Nucleotide database at NCBI (http://www.ncbi.nlm.nih.gov/nuccore/). Three clinical isolates of M. amphoriforme, one clinical isolate of Mycoplasma penetrans, and 50 clinical isolates of M. pneumoniae collected at the Bordeaux University Hospital, France, between 2006 and 2011, together with four adhesin P1 variants 2a (MP-Ho, MP-Sta, MP-79809, and MP-79692) (20) and one variant 1 (MP-4817) (21) of M. pneumoniae, were used in the study. These isolates were previously identified using a specific real-time PCR assay targeting the adhesin P1 gene (22) for M. pneumoniae and the 16S rRNA gene for M. amphoriforme (13) and using 16S rRNA gene sequencing for M. penetrans. The isolates and variants were cultured at 37°C in HG medium (7). Fifty-five clinical isolates of M. hominis collected at the Bordeaux University Hospital in 2009 and 2010 that were previously identified by culture and confirmed by a specific real-time PCR assay targeting the yidC gene (11) were cultured at 37°C in Hayflick modified broth supplemented with arginine (HA medium) (7). Six clinical isolates of U. parvum and five clinical isolates of U. urealyticum collected at the Bordeaux University Hospital and that were previously identified by a real-time PCR assay targeting the urease gene (14) were cultured at 37°C in Shepard broth (7). The clinical isolates from ruminants were grown in PPLO broth (Difco, France), supplemented as previously described (9), at 37°C and in 5% CO2. The isolates were first identified using dot immunobinding on membrane filtration (MF-dot) (9), the current method for routine strain identification at the Anses Lyon Laboratory (16) within the framework of the epidemiosurveillance network VIGIlance to MYCoplasmoses of ruminants (VIGIMYC) (23). The specimens consisted of (i) 48 M. agalactiae isolates collected between 1951 and 2009 in France and Europe from domestic small ruminants and wild ungulates with contagious agalactia, (ii) 48 M. bovis isolates collected between 1986 and 2010 worldwide from cattle with pneumonia or, to a lesser extent, mastitis (n ⫽ 5) or arthritis (n ⫽ 1), and (iii) 47 isolates from the M. mycoides cluster, mainly from M. mycoides subsp. capri (n ⫽ 19) and Mycoplasma capricolum subsp. capricolum (n ⫽ 19), with a few Mycoplasma putrefaciens (n ⫽ 4) and Mycoplasma yeatsii (n ⫽ 5) isolates, all collected from ruminants with mastitis, arthritis, or pneumonia, or in the case of the M. yeatsii strains, from asymptomatic animals. The capacity of MALDI-TOF MS to simultaneously identify two strains in mixed culture was also assessed by analyzing (i) artificially constructed mixtures of strains and (ii) a total of 73 subcultures of bovine pneumonia clinical specimens known to often contain more than one mycoplasma species. The artificial mixtures were prepared by mixing various volumes of culture in HA medium and Shepard medium for the human mycoplasmas or by mixing equal volumes of individual strain cultures after cell counting using a “most probable number” method for the ruminant species (24). The specimen subcultures had been routinely analyzed by MF-dot within the VIGIMYC network and kept frozen at ⫺20°C prior to further analysis by MALDI-TOF MS in a unique run. All analyses of the ruminant mycoplasmas were performed using a single-blind protocol. The blinding was performed in Lyon, France, where the strains were grown, and the specimens were then sent to Saint-Lô, France, for protein extraction and MALDI-TOF MS analysis. Culturing and analysis of the human mycoplasmas were performed in Bordeaux, France. Protein extraction for MALDI-TOF MS analysis. Mycoplasma pellets were obtained after centrifugation of various volumes of culture, depending on the growth capacities of the individual species. For the human mycoplasmas, 30 ml of culture of reference strain was used to generate the MSP library, with the exception for Ureaplasma spp., for which 100 ml was required. The culture volumes were the same for the identification of the human clinical isolates using MALDI-TOF MS, i.e., 30 ml for the M. pneumoniae clinical isolates and 100 ml for the Ureaplasma sp. isolates; the exception was for the M. hominis clinical isolates, for which a 1-ml culture was sufficient. For the ruminant mycoplasmas, 1 ml of culture was used for both the MSP construction and identification of clinical isolates. The.

(4) Identification of Mycoplasmas by MALDI-TOF MS. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest FIG 1 Clustering of Mycoplasma and Ureaplasma strains as a function of their 16S rRNA phylogeny (A) or their MSP dendrogram (B). (A) Phylogenetic tree showing positions of the strains used in this study. *, Strains for which a complete 16S rRNA gene sequence was not available were replaced with other strains of the same species. Only partial sequences were available for strains M. hominis H34 and M132, and these strains were consequently not included in the tree. The evolutionary history of the strains was inferred using the maximum likelihood method. The tree with the highest likelihood is shown, and Acholeplasma laidlawii PG8 was used as an outgroup. A total of 1,407 nucleotides from the 16S rRNA gene were used in the analysis. The data set was resampled 1,000 times, and the bootstrap percentage values (⬎50%) are given at the nodes. Phylogenetic groups as defined by Weisburg et al. (25) are also presented. (B) Score-oriented dendrogram of MSPs from human and ruminant mycoplasma species used to construct the mycoplasma database. The dendrogram was generated with the default settings in MALDI Biotyper (distance measure by correlation and linkage by average).. showed an overall topology that was comparable to that of the phylogenetic 16S rRNA tree defined by Weisburg et al. (25) (Fig. 1A). When using an arbitrary distance level of 900 as a cutoff, all the strains were correctly assigned to their respective phyloge-. October 2013 Volume 51 Number 10. netic groups in the dendrogram. The pneumoniae, spiroplasma, and hominis groups yielded unequivocally separated branches, whereas both the hominis and pneumoniae groups were further split into two branches. This was particularly intriguing for M.. jcm.asm.org 3317.

(5) Pereyre et al.. 3318. jcm.asm.org. clinical isolates were successfully identified by MALDI-TOF MS, with scores of ⱖ1.700. Thirty-three strains had scores of ⱖ2.000 and 13 had scores between 1.700 and 1.999. The only identification failures were due to poor growth of the strains, one of which resulted nonetheless in an accurate identification of M. agalactiae, with a score of 1.5. For M. bovis, 98% (47/48) of the analyzed isolates were correctly identified, with 36 yielding scores of ⱖ2.000 and 11 yielding scores between 1.700 and 1.999. The only strain that yielded an unsatisfactory score of 1.6 was strain 8790, which is known by both multilocus sequence typing (MLST) and 16S rRNA sequencing to have an intermediate position, between M. agalactiae and M. bovis, two highly related species of the hominis group (26). This strain was nonetheless assigned by MALDI-TOF MS to M. bovis. Forty-seven strains from the M. mycoides cluster were also analyzed, including several species and subspecies. For both M. capricolum subsp. capricolum and M. mycoides subsp. capri, 95% (18/19) of the strains were correctly assigned to their respective subspecies, with scores of ⱖ1.700. Interestingly, the only strain among the M. mycoides subsp. capri strains that yielded a score of 1.56 was not M. mycoides subsp. capri, as was suggested by its MF-dot profile; indeed, this strain was later assigned by housekeeping gene sequence analysis to the newly described species Mycoplasma feriruminatoris sp. nov. (27). The identification of the M. putrefaciens strains was congruent with the MF-dot results despite 2/4 yielding a score of ⬍1.700. In contrast, the identification scores (⬍1.300) were considered unacceptable for M. yeatsii (n ⫽ 5). Analysis of clinical specimens containing more than one mycoplasma (sub)species. Because human and ruminant clinical specimens are often simultaneously contaminated by different mycoplasma species, we assessed the capacity of MALDI-TOF MS to address mixtures of strains. When two species, M. bovis and Mycoplasma bovirhinis, U. parvum and M. hominis, or U. urealyticum and M. hominis, were artificially mixed in equivalent cellular concentrations, both species were detected by MALDI-TOF MS with an approximately equivalent score for each species. However, when the cellular ratio was unbalanced (from 1/5 to 1/100, depending on the species), the predominant species had a score of ⬎2.000, whereas the species at the lower concentration was not detected or received a score of ⬍1.300, which was considered to be uninterpretable. A total of 73 subcultures of the bovine pneumonia clinical specimens were also analyzed, 57 of which had previously been identified as a single strain by MF-dot (M. bovis, M. bovirhinis, Mycoplasma arginini, and Mycoplasma bovigenitalium) and 16 as a mixture of 2 to 3 strains from different species, including M. bovis and one or two other species, such as Mycoplasma alkalescens, M. bovirhinis, Mycoplasma canadense, M. bovigenitalium, and M. arginini. Of the 57 samples containing a single strain, 97% (55/57) were identified by MALDI-TOF MS, with scores of ⱖ1.700 and a species match that was consistent with that obtained by MF-dot. Two subcultures yielded different identification results when examined by MF-dot prior to storage at ⫺20°C (identification of M. arginini) and by MALDI-TOF MS after storage (identification of M. bovis, with scores of ⱖ1.700). This discrepancy was not the result of a wrong identification but of a modification to the ratio of the species during the storage of the strain mixtures. For the 16 specimens containing 2 to 3 species, as detected by MF-dot, M.. Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. penetrans strain GTU-54, which was isolated in a specific branch. We further examined this topological particularity by generating another MSP using a clinical strain of the M. penetrans species, namely, strain 6414. In the dendrogram, M. penetrans GTU-54 and 6414 remained grouped on a unique branch and were separated from each other by a very short distance, as would be expected for strains belonging to the same species. Identification of clinical isolates. The mycoplasma colonies are so small and often inlaid in the agar, such that in our study, direct colony deposition often failed to result in species- or genuslevel identification, with the exception of (sub)species that grew to a high density, such as M. mycoides subsp. capri and some isolates of M. hominis. Moreover, the extraction step was preferred for mycoplasma species identification because attempts to deposit the mycoplasma pellet directly onto the target plate resulted in weaker scores than when the complete extraction procedure was applied. Thus, broth culture and protein extraction were performed in all cases in this study. The required culture volume depended on the species to be identified (see below). As the broth media that are used to grow mycoplasmas contain a large concentration of proteins, the nonseeded HA, HG, Shepard, and PPLO medium batches were analyzed by MALDITOF MS. These samples were treated like the clinical isolates, as described in Materials and Methods, and the resulting spectra were compared to those in the mycoplasma and MALDI Biotyper databases. No spectral concordances were obtained, suggesting that the proteins contained in the different culture media had no significant influence on the identification of the clinical isolates. Moreover, we tested the SP4 medium (7) instead of the usual HG medium to grow the M. pneumoniae M129 reference strain before performing MALDI-TOF MS. The strain was accurately identified as M. pneumoniae M129 with a score of ⬎2.000, confirming that changing the culture medium had no influence on the identification of this species using MALDI-TOF MS. For the M. hominis species and other species growing in HA medium, preliminary tests revealed that protein extraction from a culture volume of 1 ml was sufficient to achieve species-level identification. Of the 55 M. hominis clinical isolate extracts that were spotted twice, 100% were identified to the species level, 95% (52/ 55) had a score value of ⱖ2.000 for both spots, and 5% (3/55) had only one score of ⱖ2.000 and the other score in the range 1.700 to 1.999. For M. pneumoniae and other species growing in HG medium and for the Ureaplasma species growing in Shepard medium, culture volumes of 30 ml and 100 ml, respectively, were required before protein extraction. Ten spots from the same protein extract were deposited onto the target plate. Fifty clinical isolates of M. pneumoniae and three clinical isolates of M. amphoriforme were analyzed. In all cases, at least eight spots out of 10 yielded scores of ⱖ1.700, corresponding to an accurate species-level identification. There were no misidentifications, and MALDI-TOF MS was able to distinguish M. pneumoniae from Mycoplasma genitalium, the phylogenetically closest species. Eleven clinical isolates of Ureaplasma were analyzed, consisting of six clinical isolates of U. parvum and five clinical isolates of U. urealyticum. In 100% of the cases, at least eight spots out of 10 yielded scores of ⱖ1.700, which allowed for accurate identification to the species level. For the ruminant mycoplasmas, each clinical sample was only spotted once to develop a procedure that was compatible with a routine workflow. Of the first set, 96% (46/48) of the M. agalactiae.

(6) Identification of Mycoplasmas by MALDI-TOF MS. bovis was the only component of the mixture that was identified by MALDI-TOF MS. Mycoplasma subtyping. The spectra acquired for species identification purposes were further used to assess the subtyping capacity of MALDI-TOF MS for M. pneumoniae strains, and also for M. bovis and M. agalactiae strains. Based on the analysis of the gene encoding the P1 protein, M. pneumoniae is known to comprise two subtypes (type 1 and type 2) and a few variants related to each subtype (28). Using MALDI-TOF MS, all 50 M. pneumoniae spectral profiles were accurately clustered into 2 separate groups corresponding to M. pneumoniae adhesin P1 type 1 (reference strain M. pneumoniae M129) and M. pneumoniae adhesin P1 type 2 (reference strain M. pneumoniae FH) (Fig. 2). In addition, three M. pneumoniae variant 2a strains and one variant 1 strain were accurately ranked among the type 2 and type 1 strains, respectively (Fig. 2). We also searched for an association between the MALDITOF MS results and the recently developed multilocus variablenumber tandem-repeat (VNTR) analysis (MLVA) typing method that is based on an analysis of the number of tandem repeats present at five loci of the M. pneumoniae genome (20). Although no association could be drawn because the isolates of the same MLVA. October 2013 Volume 51 Number 10. type were present in different branches of the dendrogram (Fig. 2), all MLVA types J, P, E, U, X, 29, and 31 were clustered in the adhesin P1 type 1 strains, and all MLVA types B, T, G, V, M, O, S, and H were clustered in the adhesin P1 type 2 strains. This finding was expected because a correlation between the MLVA typing results and the type of adhesin P1 gene was previously reported (20), with the former MLVA types being related to adhesin P1 type 1 and the latter MLVA types being related to adhesin P1 type 2. M. bovis and M. agalactiae are two highly related mycoplasma species that are important pathogens of cattle and small ruminants, respectively. An MLST approach was recently proposed as an unequivocal tool for strain differentiation, characterization, and molecular typing of these two species (26). To infer whether MALDI-TOF MS might be useful to type strains within each species, we generated a dendrogram using 18 strains from each species, all of which, except for the reference strains, were collected in France in different years, and from different hosts in the case of M. agalactiae (Fig. 3). In the resulting dendrogram, the M. agalactiae strains were separated into two branches that correspond to the two reference strains, namely, PG2 and 5632. For M. bovis, the strains were regrouped into two main branches according to their. jcm.asm.org 3319. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. FIG 2 Dendrogram of the MALDI-TOF MS profiles generated using MALDI Biotyper 3.0 with 50 clinical isolates of M. pneumoniae, M. pneumoniae reference strains FH and M129, four M. pneumoniae variants 2a and 1 M. pneumoniae variant 1; distance measure was set at correlation, and linkage was set at average. The adhesin P1 type and the MLVA type are given in parentheses, respectively. The reference strains M129 and FH are in bold type, and the variants are italicized. MP, M. pneumoniae..

(7) Pereyre et al.. years of isolation. In the dendrogram, we also observed that the M. agalactiae group has longer branches than the M. bovis branch, which indicates a more variable species, an observation that is in agreement with the MLST data (26). DISCUSSION. To assess the feasibility of using MALDI-TOF MS for the identification of clinically relevant mycoplasmas in both human and veterinary medicine, we first attempted to enrich an available mycoplasma database that only contained (as of the beginning of this study) one porcine mycoplasma species, Mycoplasma hyorhinis (Bruker MALDI Biotyper MSP database 3.1.2.0). For this purpose, 29 MSPs corresponding to 23 mycoplasma (sub)species were constructed, representing most of the human and ruminant pathogenic and commensal species distributed in three phylogenetic groups of the class Mollicutes, namely, spiroplasma, hominis, and pneumoniae. The inclusion of pathogenic and commensal species in this database had the two purposes of (i) being able to unequivocally exclude the presence of pathogenic species by the identification of nonpathogenic species and (ii) addressing a mixture of species in clinical specimens. For example, in ruminant respiratory specimens, M. bovis, a pathogenic mycoplasma, is frequently associated with M. bovirhinis, which is a commensal species. The MSP dendrogram resulted in a clustering of strains that was overall congruent with the 16S rRNA phylogeny, though some groups were further subdivided into several branches (Fig. 1). This observation was an important quality control for further species identification using this mycoplasma database. To limit the potential misidentification of atypical strains from species that are known to be variable, we included several strains from the. 3320. jcm.asm.org. same species in the database. For instance, three strains of M. hominis were included, as this species is known to be highly heterogeneous (29). Both M. agalactiae PG2 type strain and 5632 were acquired as well, as they are considered to be situated at each end of the genetic spectrum encountered in M. agalactiae (30). This proved successful because, for instance, several strains from wild ungulates (e.g., Capra ibex) that are known to be genetically different from domestic ruminant strains (31) were correctly identified as M. agalactiae. For M. bovis, a strain known to be pathogenic (strain 1067) (32) was used together with the type strain PG45. Lastly, within the recently reclassified M. mycoides subsp. capri taxon, the former type strain of the Mycoplasma mycoides subsp. mycoides large colony (MmmLC) biotype, namely, GM12, was included in addition to the PG3 type strain. Generating several MSPs per species is also a convenient step for subtyping strains once they have been assigned to a species (see below). While this study was being conducted, Bruker released an update of their own database (MALDI Biotyper MSP database 3.3.1.0, April 2012), which included a total of 10 species of the Mycoplasma genus. However, only six ruminant mycoplasma species were added, none of which are from the M. mycoides cluster, and no human mycoplasma species or strains from the Ureaplasma genus were included. The mycoplasmal cultivation and sample preparation were optimized to be compatible with the laboratory diagnostic workflow. The direct deposition of a colony or a cell pellet on the target plate was shown to occasionally yield species-level identification, though with scores weaker than those obtained with the protein extraction process. As a consequence, as was already suggested for other bacteria (1), we chose to apply the protein extraction protocol to increase the rates of identification. This protocol relies on a. Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. FIG 3 Dendrogram of MALDI-TOF MS profiles generated using MALDI Biotyper 3.0 with 36 clinical isolates of M. agalactiae and M. bovis. The M. agalactiae strains are designated Maga, followed by the strain number, the year of isolation, and the host species. The M. bovis strains are designated Mbov, followed by the strain and the year of isolation in parentheses..

(8) Identification of Mycoplasmas by MALDI-TOF MS. October 2013 Volume 51 Number 10. ture of species by MALDI-TOF MS because the precultivation step results in a high predominance of the cultured species. However, such discrimination is of interest for ruminant species that grow in the same medium, as we previously reported that ⬎10% of clinical specimens might contain more than one mycoplasma (sub)species (23). However, in the 16 specimens containing two to three ruminant species, MALDI-TOF MS only detected M. bovis, the clinically relevant species, which may have been favored to the detriment of other species during the storage phase at ⫺20°C. Another potential explanation for these discrepancies between MF-dot and MALDI-TOF MS identification techniques might be based on the slightly better sensitivity of MF-dot (from 2 ⫻ 104 to 8 ⫻ 106 mycoplasma organisms per well [9]) versus MALDI-TOF MS (106 CFU per spot [33]), which can result in the codetection of some nonpredominant species when using MF-dot. In this study, different identification results were obtained for two clinical specimens that were not considered to be mixtures based on MF-dot results, e.g., M. arginini by MF-dot and M. bovis by MALDI-TOF MS. These results might point toward another mixture of strains in which M. bovis was overgrown by M. arginini when the MF-dot was performed but for which the species ratio was modified by a long period of storage at ⫺20°C prior to MALDI-TOF MS identification. In conclusion, as already shown by Stevenson et al. (33), not all organisms that are present in a polymicrobial sample can be reliably detected by MALDI-TOF MS. MALDI-TOF MS allowed for the identification of Ureaplasma spp. at the species level, which is generally not achieved with routine laboratory diagnostic methods. Indeed, the former U. urealyticum species consisted of a heterogeneous species comprising 2 biovars and 14 serovars. This taxon was split 10 years ago into two distinct species, U. parvum and U. urealyticum, corresponding to the former biovar 1 (including 4 serovars) and biovar 2 (including 10 serovars), respectively (37). The distinction between the species requires either antibody-based phenotyping methods, which are often inconclusive because of multiple cross-reactions, or more accurate molecular techniques (14, 38). Although an additional overnight incubation was necessary to obtain a 100-ml culture volume, MALDI-TOF MS allowed for easy identification of Ureaplasma spp. to the species level, which is a benefit of the clinical use of MALDI-TOF MS because several studies have reported U. urealyticum to be more pathogenic than U. parvum (39, 40). MALDI-TOF MS also proved to accurately distinguish species that are known to be closely related, M. pneumoniae, M. genitalium, and M. amphoriforme, which grow in the same HG medium, and M. bovis and M. agalactiae, which used to be classified as two subspecies of the same species prior to being separated into two different species based on serological, DNA-DNA reassociation experiments (41), and 16S rRNA sequence data (42). Furthermore, within individual species, the dendrograms generated from the MALDI-TOF MS spectra achieved epidemiologically relevant strain clustering. For instance, the M. agalactiae strains were separated into two branches that correspond to the two reference strains, namely, PG2 and 5632, which are described as representing each end of the genetic spectrum encountered in M. agalactiae (30). The strains from wild ungulates, namely, Capra ibex or chamois, interestingly were grouped together, as was previously reported with the use of partial sequencing of a housekeeping gene (31). For M. bovis, the strains were regrouped into two main branches that correspond to recent and old strains, a subclustering that is consistent with the recent data obtained in our group by. jcm.asm.org 3321. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. series of centrifugation and resuspension steps that are, in our opinion, compatible with routine usage. An increased number of washing steps performed on the mycoplasma pellet prior to extraction improved the spectral quality, and this was mainly due to the removal of the strong protein background generated by the yeast extract and horse serum, two major components of mycoplasma growth media. Nonetheless, increasing the number of washing steps also resulted in a decrease in the quantity of the proteins that were contained in the final extract. For clinical identification, a 1-ml culture was sufficient to obtain interpretable spectra for most species, except for those growing in HG medium, such as M. pneumoniae, or in Shepard medium, such as Ureaplasma spp., for which 30 ml and 100 ml of culture, respectively, were required. Handling such volumes might not be compatible with a routine clinical workflow; however, attempts to reduce these volumes while obtaining similar results failed. These results are consistent with the cell densities of the individual cultures. For instance, M. hominis, one of the less fastidious human mycoplasmas, typically reaches concentrations of up to 108 to 109 color-changing units (CCU)/ml at the mid-log phase in HA medium, whereas M. pneumoniae and Ureaplasma spp. only yield concentrations of 107 to 108 CCU/ml and 106 to 107 CCU/ml, respectively. Most of the ruminant species analyzed in this study reached a concentration of 108 to 1010 CFU/ml at the stationary phase, and Stevenson et al. (33) determined that excellent spectra were obtained when a minimum of 106 CFU was spotted onto the target plate, which is approximately what we obtained when starting from 1 ml of a 108-CFU/ml culture. Bruker Daltonics recommends scores of ⱖ1.700 and ⱖ2.000 for genus- and species-level identifications, respectively. However, as has previously been demonstrated for other bacterial species (3, 34, 35, 36), our results strongly advocate a reduction of the acceptable scores for mycoplasma species identification to ⱖ1.700. With these parameters, the rate of species-level identification was 100% with no misidentifications, even for the low-celldensity species, i.e., Ureaplasma spp. and M. pneumoniae. Furthermore, several subspecies of the complex M. mycoides cluster that are known to be closely related were unequivocally identified. Additionally, based on the high rate of identification to the (sub) species level, a reduction in the number of spots that were deposited on the target plate from the same isolate was considered when appropriate. For species that grow in HA medium, such as M. hominis, one spot can be utilized for routine diagnosis because in our study, it yielded a score of ⱖ1.700 in all cases. In contrast, 10 spots were utilized for more fastidious species that grow in HG or Shepard medium. When considering the results that were obtained using the first three spots for the 50 M. pneumoniae isolates and 11 Ureaplasma isolates, identification to the species level with a score of ⱖ1.700 for two out of these three spots was obtained in 98% of cases (60/61), suggesting that three spots are sufficient for these organisms. For the ruminant mycoplasmas, a single spot was deposited, leading to a correct identification in 95 to 98% of cases with a score of ⬎1.700. In cases of a misidentification or a score of ⬍1.700, we recommend either relaunching the analysis with several spots or increasing the quantity of starting material. We further demonstrated that MALDI-TOF MS can accommodate in vitro-reconstituted mixtures of species up to a certain ratio. Regarding human urogenital mycoplasma species, it must be noted that Ureaplasma spp. and M. hominis do not grow in the same culture medium, which reduces the need to detect this mix-.

(9) Pereyre et al.. 13.. 14. 15. 16.. 17. 18.. 19.. ACKNOWLEDGMENTS We thank Marc Bonneu and Jean-William Dupuy from the genomic platform of the University Bordeaux Segalen for their advice. We are grateful to all VIGIMYC members, François Poumarat, and the technical staff in Lyon responsible for managing the collection of the ruminant mycoplasmas strains. We thank Marie Gardette for technical assistance.. 20.. REFERENCES. 22.. 1. Croxatto A, Prod’hom G, Greub G. 2011. Applications of MALDI-TOF mass spectrometry in clinical diagnostic microbiology. FEMS Microbiol. Rev. 36:380 – 407. 2. Ford BA, Burnham CA. 2013. Optimization of routine identification of clinically relevant Gram-negative bacteria by use of matrix-assisted laser desorption ionization–time of flight mass spectrometry and the Bruker Biotyper. J. Clin. Microbiol. 51:1412–1420. 3. McElvania Tekippe E, Shuey S, Winkler DW, Butler MA, Burnham CA. 2013. 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Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro- and micro-events that are shaping mycoplasma diversity. BMC Genomics 11:86. doi:10.1186/1471 -2164-11-86. Tardy F, Baranowski E, Nouvel LX, Mick V, Manso-Silvàn L, Thiaucourt F, Thébault P, Breton M, Sirand-Pugnet P, Blanchard A, Garnier A, Gibert P, Game Y, Poumarat F, Citti C. 2012. Emergence of atypical Mycoplasma agalactiae strains harboring a new prophage and as-. Journal of Clinical Microbiology. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. MLST and MLVA (C. A. Becker, personal communication). Although these preliminary typing results are promising, further studies that include a larger number of isolates are needed to confirm these findings. Moreover, MALDI-TOF MS allowed for not only the identification of M. pneumoniae isolates but also their adhesin P1 typing in a single step, without the need of additional molecular typing techniques (28, 43, 44). However, no direct correlation was found between MALDI-TOF MS and MLVA typing. In this study, we developed a mycoplasma spectral database of human and ruminant mycoplasmas that proved to be reliable for the identification of clinical isolates. A high concordance was found between the MALDI-TOF MS species identification and species identification using biochemical, antigenic, or molecular methods. MALDI-TOF MS is a rapid, reliable, and cost-effective method, particularly for the routine identification of M. hominis and ruminant mycoplasmas that grow to a high cell density, and it might replace conventional identification methods in the future. Moreover, MALDI-TOF MS with no further optimization proved to be useful for the subtyping of several mycoplasma species and may be promising for other typing developments..

(10) Identification of Mycoplasmas by MALDI-TOF MS. 32.. 33.. 34.. 35.. 37.. October 2013 Volume 51 Number 10. 38.. 39.. 40.. 41.. 42.. 43.. 44.. and emended description of Ureaplasma urealyticum (Shepard et al. 1974) Robertson et al. 2001. Int. J. Syst. Evol. Microbiol. 52(Pt 2):587–597. Xiao L, Paralanov V, Glass JI, Duffy LB, Robertson JA, Cassell GH, Chen Y, Waites KB. 2011. Extensive horizontal gene transfer in ureaplasmas from humans questions the utility of serotyping for diagnostic purposes. J. Clin. Microbiol. 49:2818 –2826. Deguchi T, Yoshida T, Miyazawa T, Yasuda M, Tamaki M, Ishiko H, Maeda SI. 2004. Association of Ureaplasma urealyticum (biovar 2) with nongonococcal urethritis. Sex. Transm. Dis. 31:192–195. Povlsen K, Bjørnelius E, Lidbrink P, Lind I. 2002. Relationship of Ureaplasma urealyticum biovar 2 to nongonococcal urethritis. Eur. J. Clin. Microbiol. Infect. Dis. 21:97–101. Askaa G, Erno H. 1976. Elevation of Mycoplasma agalactiae subsp. bovis to species rank: Mycoplasma bovis (Hale et al.) comb. nov. Int. J. Syst. Bacteriol. 323–325. Pettersson B, Uhlén M, Johansson KE. 1996. Phylogeny of some mycoplasmas from ruminants based on 16S rRNA sequences and definition of a new cluster within the hominis group. Int. J. Syst. Bacteriol. 46:1093– 1098. Schwartz SB, Thurman KA, Mitchell SL, Wolff BJ, Winchell JM. 2009. Genotyping of Mycoplasma pneumoniae isolates using real-time PCR and high-resolution melt analysis. Clin. Microbiol. Infect. 15:756 –762. Spuesens EB, Hoogenboezem T, Sluijter M, Hartwig NG, van Rossum AM, Vink C. 2010. Macrolide resistance determination and molecular typing of Mycoplasma pneumoniae by pyrosequencing. J. Microbiol. Methods 82:214 –222.. jcm.asm.org 3323. Downloaded from http://jcm.asm.org/ on May 16, 2020 by guest. 36.. sociated with an alpine wild ungulate mortality episode. Appl. Environ. Microbiol. 78:4659 – 4668. Hermeyer K, Buchenau I, Thomasmeyer A, Baum B, Spergser J, Rosengarten R, Hewicker-Trautwein M. 2012. Chronic pneumonia in calves after experimental infection with Mycoplasma bovis strain 1067: characterization of lung pathology, persistence of variable surface protein antigens and local immune response. Acta Vet. Scand. 54:9. doi:10.1186/1751 -0147-54-9. Stevenson LG, Drake SK, Murray PR. 2010. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization–time of flight mass spectrometry. J. Clin. Microbiol. 48: 444 – 447. Bizzini A, Jaton K, Romo D, Bille J, Prod’hom G, Greub G. 2011. Matrix-assisted laser desorption ionization–time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains. J. Clin. Microbiol. 49:693– 696. Alatoom AA, Cazanave CJ, Cunningham SA, Ihde SM, Patel R. 2012. Identification of non-diphtheriae Corynebacterium by use of matrixassisted laser desorption ionization–time of flight mass spectrometry. J. Clin. Microbiol. 50:160 –163. Alatoom AA, Cunningham SA, Ihde SM, Mandrekar J, Patel R. 2011. Comparison of direct colony method versus extraction method for identification of gram-positive cocci by use of Bruker Biotyper matrix-assisted laser desorption ionization–time of flight mass spectrometry. J. Clin. Microbiol. 49:2868 –2873. Robertson JA, Stemke GW, Davis JW, Jr, Harasawa R, Thirkell D, Kong F, Shepard MC, Ford DK. 2002. Proposal of Ureaplasma parvum sp. nov..

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