Diagnostic Microbiology and Infectious Disease 85 (2016) 9–11
Contents lists available at ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
MALDI-TOF mass spectrometry for differentiation between Streptococcus pneumoniae and Streptococcus pseudopneumoniae Joffrey van Prehn a,b,⁎, Suzanne Q. van Veen b, Jacqueline J.G. Schelfaut c, Els Wessels c a b c
Department of Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands Department of Medical Microbiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
a r t i c l e
i n f o
Article history: Received 9 November 2015 Received in revised form 21 December 2015 Accepted 21 January 2016 Available online 22 January 2016 Keywords: Streptococcus Mitis group MALDI-TOF Mass spectrometry PCR Diagnostic accuracy
a b s t r a c t We compared the Vitek MS and Microflex MALDI-TOF mass spectrometry platform for species differentiation within the Streptococcus mitis group with PCR assays targeted at lytA, Spn9802, and recA as reference standard. The Vitek MS correctly identified 10/11 Streptococcus pneumoniae, 13/13 Streptococcus pseudopneumoniae, and 12/13 S. mitis/oralis. The Microflex correctly identified 9/11 S. pneumoniae, 0/13 S. pseudopneumoniae, and 13/13 S. mitis/oralis. MALDI-TOF is a powerful tool for species determination within the mitis group. Diagnostic accuracy varies depending on platform and database used. © 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
It is clinically relevant to distinguish Streptococcus pneumoniae from other less virulent, members of the viridans group streptococci (VGS). Accurate species determination within the VGS and more specifically within the mitis subgroup is traditionally difficult. To overcome this problem, PCR assays targeting virulence genes and fragments, such as the autolysin gene (lytA), the pneumolysin gene (ply), and the Spn9802 DNA fragment, have been designed. However, also nonpneumococcal species of the VGS may harbor lytA and ply (Whatmore et al., 2000), thereby affecting the specificity of these assays. Sequence analysis of the 16S rRNA gene also has limited discriminatory power within the VGS, as Streptococcus mitis, Streptococcus oralis, and S. pneumoniae exhibit more than 99% sequence homology (Kawamura et al., 1995). Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry shows promising results for differentiation of species within the mitis group (Angeletti et al., 2015), but further exploration and validation are needed. To complicate the diagnostic challenges within the VGS, in 2004, a new species within the VGS that closely resembles S. pneumoniae was described and designated as Streptococcus pseudopneumoniae. To this end, a real-time PCR assay for the specific detection of S. pseudopneumoniae has been developed (Zbinden et al., 2011; Sistek et al., 2012). To gain further insight in the clinical relevance of S. pseudopneumoniae, it is important to have accurate diagnostic methods available.
⁎ Corresponding author. Tel.: +31-20-44-40488. E-mail address:
[email protected] (J. van Prehn).
We evaluated the ability of 2 MALDI-TOF mass spectrometry platforms for species differentiation within the mitis subgroup. A panel consisting of 29 clinical and 8 reference isolates was tested with both the Biotyper and Vitek MS MALDI-TOF mass spectrometry platforms. The reference strains used included 2 S. pneumoniae (ATCC 6305 and ATCC 49619), 2 S. pseudopneumoniae (CCUG 48465 and CCUG 49455), 2 S. mitis (LMG 14552 and LMG 14557), and 2 S. oralis (LMG 14532 of 14533). As a gold standard, we combined real-time PCR assays targeting lytA, recA, and Spn9802, which were adapted from methods described by Sistek et al. (2012), Carvalho Mda et al. (2007), and Suzuki et al. (2005). The assays were performed as described by Wessels et al. (2012). Instead of 50-μL reactions, 10-μL 0.5 McFarland solution was added to 15-μL PCR mix, resulting in 25-μL reactions. No DNA isolation was performed. The PCR assays targeting the lytA and recA genes are specific for S. pneumoniae and S. pseudopneumoniae, respectively. The PCR assay targeting the Spn9802 fragment detects both S. pneumoniae and S. pseudopneumoniae but no other VGS. Hence, strains that tested negative in all 3 PCR assays were designated S. mitis/oralis. MALDI-TOF mass spectrometry with the Microflex platform (Bruker Daltonics, Bremen, Germany) was performed as previously described (van Veen et al., 2010). Single colonies were transferred in duplicate onto a target plate and overlaid with matrix solution. FlexControl software (version 3.4) was used for measurements. The spectra were analyzed using BioTyper software (version 3.1.66; Bruker) and database MBT DB-5627. An identification score of ≥2.0 was considered a reliable genus and species identification. Results that indicated S. oralis, S. mitis, or other (nonpneumococcus or pseudopneumococcus) species
http://dx.doi.org/10.1016/j.diagmicrobio.2016.01.012 0732-8893/© 2016 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
10
J. van Prehn et al. / Diagnostic Microbiology and Infectious Disease 85 (2016) 9–11
of the mitis group were classified into the S. mitis/oralis group for analysis. Discordant results of the first best match and second best match or inconsistency of the duplicate determination (unless both in the S. mitis/oralis group) were classified as an ambiguous result. Mass spectrometry with the Vitek MS platform (bioMérieux, Marcy l’Etiole, France) was performed using the following software and database: VitekMS ACQ 1.4.2b, VitekMS Prep 2.3.3, and Myla 3.2.0. Single colonies were transferred onto a target plate, and a single measurement was performed. An identification score of ≥95% was considered to be a reliable identification at the species level. If no identification could be assigned, a second run was performed. If this second run also failed to assign a species, then the strain was regarded as unidentified for analysis. The results of the determinations with real-time PCR, the Vitek MS, and the Microflex platform are shown in Table 1. In total, the panel included 11 pneumococci, 13 pseudopneumococci, and 13 S. mitis/oralis. The sensitivity and specificity of the 2 MALDI-TOF systems for detection of the separate species are shown in Table 2. Using the Vitek MS platform, 1 S. mitis/oralis strain could not be identified, and 1 S. pneumoniae strain was incorrectly identified as S. mitis/oralis group. All other strains were identified correctly. Using the Microflex platform, all S. mitis/oralis and 9 of the 11 S. pneumoniae in the panel were correctly identified. However, none of the S. pseudopneumoniae in the panel was correctly identified. We found that mass spectrometry has the ability to adequately distinguish relevant species within the mitis group streptococci. However, we observed that the Microflex platform could not differentiate between pneumococci and pseudopneumococci, whereas the Vitek MS platform could. This is likely attributable to the use of different databases and/or different determination algorithms. The database used by the Microflex platform contains 30 S. pneumoniae isolates and only 1 S. pseudopneumoniae isolate. The Microflex platform matches the spectrometry pattern of the isolates tested to the pattern of a best matching single isolate pattern (multiple reference isolates of most species are in the database), whereas the Vitek MS platform matches the spectrometry pattern to a composite pattern of a species (which is composed of multiple patterns of several reference isolates of a species). Difference in mass resolution between the mass spectrometers may also be of relevance: the Vitek MS has a larger tube and possibly more detailed spectra and thereby more accuracy might be obtained. This could be evaluated further using larger Biotyper platforms. Although we report on a relative small sample size, we actually have one of the largest numbers of pseudopneumococci studied by mass spectrometry (Angeletti et al., 2015; Werno et al., 2012). Indeed, other groups have studied MALDI-TOF determination of larger streptococci panels, but theses panels included far less S. pseudopneumoniae (Dubois et al., 2013). Other recent studies that compared MALDI-TOF platforms did not specifically focus on the mitis group (Martiny et al., 2012; Deak et al., 2015). Furthermore, to our knowledge, our study is only the second study that investigates a direct comparison between 2 MALDI-TOF platforms for discrimination between pneumococci and pseudopneumococci (Angeletti et al., 2015). Despite the small number
Table 1 Distribution of determinations with real-time PCR, the Vitek MS, and the Microflex platform. n
Real-time PCR
Vitek MS
Microflex
8 2 1 3 2 7 1 12 1
S. pneumoniae S. pneumoniae S. pneumoniae S. pseudopneumoniae S. pseudopneumoniae S. pseudopneumoniae S. pseudopneumoniae S. mitis/oralis S. mitis/oralis
S. pneumoniae S. pneumoniae S. mitis/oralis S. pseudopneumoniae S. pseudopneumoniae S. pseudopneumoniae S. pseudopneumoniae S. mitis/oralis No ID
S. pneumoniae Ambiguous S. pneumoniae S. pneumoniae S. mitis/oralis Ambiguous No ID S. mitis/oralis S. mitis/oralis
Table 2 The sensitivity and specificity of the 2 MALDI-TOF systems. S. pneumoniae
S. pseudopneumoniae
S. mitis/oralis
TP FP Sens Spec (n) (n)
TP FP Sens (n) (n)
TP FP Sens (n) (n)
Vitek MS 10 Microflex 9 Real-time 11 PCR
0 3 n/ a
91% 82% n/a
100% 13 83% 0 n/a 13
0 0 n/ a
Spec
100% 100% 12 0% 100% 13 n/a n/a 13
1 2 n/ a
Spec
92% 96% 100% 86% n/a n/a
TP = true positives; FN = false negatives; Sens = sensitivity; Spec = specificity; n/ a = not applicable. The numbers of true positives, false negatives, sensitivity, and specificity of the 2 MALDITOF systems for detection of different species of the mitis group streptococci are shown. For PCR, no false positives, sensitivity, and specificity are calculated as PCR (targeted at Spn9802, lytA, and recA) was the reference standard.
of isolates, we feel that our study highlights differences in diagnostic accuracy between different MALDI-TOF platforms and databases for particular bacterial species. It is of interest that Angeletti et al. (2015) recently published a paper with similar results in which the Microflex never identified S. pseudopneumoniae. However, in their study, only 2 of 17 pseudopneumococci were identified by the Vitek MS. This difference most likely can be explained by the use of different database or software version, although the use of a different molecular reference standard (rpoB gene sequencing) or clonal differences in S. pseudopneumoniae strains used may also play a role. In conclusion, mass spectrometry can be a powerful tool for bacterial species determination within the mitis group streptococci. Clinical microbiologist and researchers should be aware that different MALDITOF platforms rely on different databases and algorithms for species determination and that subsequent differences in diagnostic accuracy seem to exist. Continuous improvement and evaluation of mass spectrometry databases are recommended. Conflicts of interest No conflict of interest. Funding No specific funds or grants were used for this study. Acknowledgments The authors would like to thank Franka M. Baalbergen for her technical assistance. References Angeletti S, Dicuonzo G, Avola A, Crea F, Dedej E, Vailati F, et al. Viridans group streptococci clinical isolates: MALDI-TOF mass spectrometry versus gene sequence-based identification. PLoS 2015;10:e0120502. http://dx.doi.org/10.1371/journal.pone.0120502. Carvalho Mda G, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW, et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol 2007;45:2460–6. Deak E, Charlton CL, Bobenchik AM, Miller SA, Pollet S, McHardy IH, et al. Comparison of the Vitek MS and Bruker Microflex LT MALDI-TOF MS platforms for routine identification of commonly isolated bacteria and yeast in the clinical microbiology laboratory. Diagn Microbiol Infect Dis 2015;81:27–33. Dubois D, Segonds C, Prere MF, Marty N, Oswald E. Identification of clinical Streptococcus pneumoniae isolates among other alpha and nonhemolytic streptococci by use of the Vitek MS matrix-assisted laser desorption ionization–time of flight mass spectrometry system. J Clin Microbiol 2013;51:1861–7. Kawamura Y, Hou XG, Sultana F, Miura H, Ezaki T. Determination of 16S rRNA sequences of Streptococcus mitis and Streptococcus gordonii and phylogenetic relationships among members of the genus Streptococcus. Int J Syst Bacteriol 1995;45:406–8. Martiny D, Busson L, Wybo I, El Haj RA, Dediste A, Vandenberg O. Comparison of the Microflex LT and Vitek MS systems for routine identification of bacteria by matrixassisted laser desorption ionization–time of flight mass spectrometry. J Clin Microbiol 2012;50:1313–25.
J. van Prehn et al. / Diagnostic Microbiology and Infectious Disease 85 (2016) 9–11 Sistek V, Boissinot M, Boudreau DK, Huletsky A, Picard FJ, Bergeron MG. Development of a real-time PCR assay for the specific detection and identification of Streptococcus pseudopneumoniae using the recA gene. Clin Microbiol Infect 2012; 18:1089–96. Suzuki N, Seki M, Nakano Y, Kiyoura Y, Maeno M, Yamashita Y. Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization. J Clin Microbiol 2005;43:4528–34. van Veen SQ, Claas EC, Kuijper EJ. High-throughput identification of bacteria and yeast by matrix-assisted laser desorption ionization-time of flight mass spectrometry in conventional medical microbiology laboratories. J Clin Microbiol 2010;48:900–7. Werno AM, Christner M, Anderson TP, Murdoch DR. Differentiation of Streptococcus pneumoniae from nonpneumococcal streptococci of the Streptococcus mitis group by
11
matrix-assisted laser desorption ionization-time of flight mass spectrometry. J Clin Microbiol 2012;50:2863–7. Wessels E, Schelfaut JJ, Bernards AT, Claas EC. Evaluation of several biochemical and molecular techniques for identification of Streptococcus pneumoniae and Streptococcus pseudopneumoniae and their detection in respiratory samples. J Clin Microbiol 2012;50:1171–7. Whatmore AM, Efstratiou A, Pickerill AP, Broughton K, Woodard G, Sturgeon D, et al. Genetic relationships between clinical isolates of Streptococcus pneumoniae, Streptococcus oralis, and Streptococcus mitis: characterization of “atypical” pneumococci and organisms allied to S. mitis harboring S. pneumoniae virulence factor-encoding genes. Infect Immun 2000;68:1374–82. Zbinden A, Köhler N, Bloemberg GV. RecA-based PCR assay for accurate differentiation of Streptococcus pneumoniae from other viridans streptococci. J Clin Microbiol 2011;49:523–7.
