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  • Original article
  • Open Access

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) score algorithm for identification of Gordonia species

  • 1, 2, 3Email author,
  • 1, 3,
  • 1,
  • 1, 2, 3 and
  • 1, 3
AMB Express20188:121

https://doi.org/10.1186/s13568-018-0650-z

  • Received: 27 May 2018
  • Accepted: 18 July 2018
  • Published:

Abstract

Gordonia species differentiation is a tedious task. Herein, Gordonia identification was performed according to the standard Bruker score system and a recently proposed score for Gram positive rods identification (≥ 1.5 genus level and ≥ 1.7 species level). New scores significantly improved the identification at genus and species level.

Keywords

  • Gordonia species identification
  • MALDI-TOF MS
  • Molecular technology
  • Discriminatory peaks

Introduction

The bacterial genus Gordonia currently includes 39 different species (Sowani et al. 2017). This genus is particularly known for its biotechnological applications and it is also notable for being an opportunistic human pathogen, causing a broad spectrum of diseases in healthy as well as in immunocompromised individuals (Ramanan et al. 2013).

The phenotypic identification of Gordonia genus species has been primarily based on biochemical tests (Verma et al. 2006). However, this methodology is time-consuming and frequently leads to misidentifications. Besides, new Gordonia species are continuously being described and biochemical tests commercially available for Gram-positive rods identification are not able to adequately classify all Gordonia at the species level. Therefore, gene sequencing has become the best technique to ensure a proper identification (Hsueh et al. 2014).

Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is an automated method recently implemented as a tool for bacterial identification in clinical microbiology laboratories. Based on poor laboratory results in the identification of Gordonia by MALDI-TOF MS some authors have suggested changing the cut-off points for Gordonia identification at the genus and species level, the proposed scores being ≥ 1.5 and ≥ 1.7 respectively (Rodríguez-Lozano et al. 2016; Barberis et al. 2014; Bizzini et al. 2010). This value system differs from the manufacturer’s recommendations which are score values ≥ 2.0 for identification at species level, between 2.0 and ≥ 1.7 at genus level and being a score of ≤ 1.7 considered as unreliable identification.

The aims of this work were to assess the implementation of MALDI-TOF MS for Gordonia species identification compared with 16S rRNA gene sequencing as the gold standard and to try to define discriminatory peaks for Gordonia species differentiation.

Materials and methods

Between 1998 and 2015, 24 clinical isolates belonging to 21 different patients were identified as Gordonia at the Microbiology Department of Hospital Universitario Donostia, San Sebastian (Basque Country, Spain).Only one isolate per patient was considered for the study. Presumptive identification was established by the growth of characteristic salmon to orange colonies, coryneform Gram-positive rods appearance and non- or slightly-acid-fast bacilli. Biochemical identification was done using the API Coryne system (bioMérieux, France) which contained 20 miniaturized biochemical tests. Definitive species identification was established by sequencing a fragment of 1188 bp of the 16S rRNA gene using primers 5F (TGGAGAGTTTGATCCTGGCTAG) and 1193R (ACGTCATCCCCGCTTCCTT). A > 99% sequence identity with the sequences of Gordonia species available at GenBank using BLAST software (http://www.ncbi.nlm.nih.gov) was used as criteria for species identification.

Overall, 16 Gordonia sputi, 4 Gordonia otitidis and 1 Gordonia bronchialis isolates were identified in sputum (n = 15), urine (n = 1) and blood (n = 5) samples from 21 patients (15 men, 6 women). In some patients the pathogenic role of Gordonia isolates could not be unequivocally ascertained, especially in those cases where the microorganism was isolated from sputum, given that Gordonia respiratory infections have been rarely described in literature (Brust et al. 2009) and patients suffered from other multiples illnesses.

Bacterial extracts for MALDI-TOF MS identification (Bruker, Daltonics, Germany) was carried out according to the manufacturer’s recommendations and results interpretation was done according to the Bruker score system and the proposed new scores. Measurements were carried out in triplicate and the average of the three results was used for analysis (Bizzini et al. 2010). Since Gordonia have been previously classified using biochemical tests as Rhodococcus (Tindall and Euzéby 2001; Lam et al. 2014), six Rhodococcus equi clinical isolates were also tested by MALDI-TOF to assess the possibility of misidentification with Gordonia species.

To determine discriminatory peaks for species identification, besides the spectra obtained for the 21 clinical isolates, the reference spectra of the following Gordonia reference strains available at the Bruker library were used: Gordonia aichiensis, Gordonia alkanivorans, Gordonia australis, Gordonia bronchialis (n = 2), Gordonia rubripertincta (n = 11), and Gordonia sputi (n = 4). All peak data were analyzed by the Pearson´s correlation matrix test performed with SPSS V24 software. Differences in the results obtained for Gordonia species identification using the two different score criteria were evaluated by the Fisher exact test using GraphPad Instat version 3.05 software (GraphPad Software Inc., La Jolla, CA, USA).

Results

Using the 16S rRNA gene sequencing as standard and comparing the manufacturer’s and the new described score system for Gordonia identification, 10 isolates showing an unreliable identification (score < 1.700) were correctly identified at the genus level (Table 1) with the new system (p < 0.001) and 9 isolates correctly identified at genus level could be identified at the species level (p = 0.006). Two isolates were correctly identified by both methods at species level.
Table 1

Gordonia species identification by 16S rRNA gene sequencing and correlation with MALDI-TOF MS

Laboratory coding number

Molecular identification (16S rRNA gene)

GenBank accession number

Score

MALDI-TOF MS first species in the identification ranking list

Identification according to

Manufacturer’ scores

New scores

65206307

G. bronchialis

MH371019

1.899

G. bronchialis

Genus level

Species level

10999451

G. sputi

MH371338

1.864

G. sputi

Genus level

Species level

226573

G. sputi

MH368686

1.659

G. sputi

Unreliable identification

Genus level

228715

G. sputi

MH371337

1.633

G. rubripertincta

Unreliable identification

Genus level

234219

G. sputi

MH370805

2.05

G. sputi

Species level

Species level

239340

G. sputi

MH370806

1.742

G. sputi

Genus level

Species level

240919

G. sputi

MH370807

1.679

G. sputi

Unreliable identification

Genus level

241080

G. sputi

MH368687

1.703

G. sputi

Genus level

Species level

241362

G. sputi

MH370809

1.867

G. sputi

Genus level

Species level

241859

G. sputi

MH368688

2.12

G. sputi

Species level

Species level

241867

G. sputi

MH368689

1.549

G. rubripertincta

Unreliable identification

Genus level

241963

G. sputi

MH368690

1.72

G. sputi

Genus level

Species level

243347

G. sputi

MH370808

1.765

G. sputi

Genus level

Species level

243815

G. sputi

MH368691

1.768

G. sputi

Genus level

Species level

243837

G. sputi

MH368692

1.695

G. sputi

Unreliable identification

Genus level

245108

G. sputi

MH368693

1.64

G. sputi

Unreliable identification

Genus level

245419

G. sputi

MH368694

1.715

G. sputi

Genus level

Species level

240706

G. otitidis

MH371196

1.507

G. rubripertincta

Unreliable identification

Genus level

243669

G. otitidis

MH370799

1.674

G. aichiensis

Unreliable identification

Genus level

243814

G. otitidis

MH370800

1.638

G. rubripertincta

Unreliable identification

Genus level

245663

G. otitidis

MH370801

1.517

G. rubripertincta

Unreliable identification

Genus level

The four G. otitidis isolates were misidentified probably because of the absence of any G. otitidis reference isolate in the Bruker database.

None of the 6 R. equi isolates was misidentified as Gordonia although in only one case the MALDI-TOF MS identified the isolate as R. equi with a score value of 2.087. In the other five isolates the identification score was ≤ 1.2 not given a reliable identification despite the good peak profiles obtained.

To define discriminatory peaks for species identification, the spectra of 6 (15.4%) of the 39 different Gordonia species described up to date were employed. The Pearson correlation coefficient showed that most species were closely related with the exception of G. rubripertincta (r = 0.363) and G. alkanivorans (r = − 0.069). The spectra of G. sputi and G. otitidis clinical isolates were closely related (r = 0.946) and two discriminatory peaks at 3332.8 and 4117.5 m/z could be established. The presence of one of those peaks or both was enough to discriminate all G. sputi and G. otitidis isolates from G. aichiensis, G. alkanivorans, G. australis, G. bronchialis, and G. rubripertincta but not between them.

Discussion

Of the 39 Gordonia species described to date only 10 have been identified as opportunistic pathogens in humans capable to cause a wide range of diseases ranging from primary cutaneous infections or lung infections to bacteremia or recurrent breast abscesses in healthy individuals as well as in immunocompromised patients (Sowani et al. 2017; Rodríguez-Lozano et al. 2016). Gordonia is difficult to identify using conventional biochemical tests as reflected in some reports (Tindall and Euzéby 2001; Lam et al. 2014) where all Gordonia isolates were misidentified as Rhodococcus species. To date, sequencing of the 16S rRNA gene has become the most reliable technique for Gordonia species identification (Bizzini et al. 2010). However, sequencing results are not generated instantly and are not available in all laboratories.

Recently, MALDI-TOF MS based system has emerged as new tool with the possibility to identify most of the microorganisms causing human infections (Lam et al. 2014). Different studies evaluated the precision of MALDI-TOF MS for Gordonia species identification with not very promising results (Hsueh et al. 2014; Rodríguez-Lozano et al. 2016; Barberis et al. 2014; Bizzini et al. 2010; Lai et al. 2010). In fact, based on previous studies (Rodríguez-Lozano et al. 2016; Barberis et al. 2014; Bartolomé-Álvarez et al. 2016), a lower score cut–off for an accurate identification at species and genus level was suggested. In our study, a lower score significantly increased the correct identification from 10 to 20 isolates at genus level and from 2 to 11 isolates at species level without affecting the specificity.

Our work, although with a limited number of different Gordonia species, confirmed that a lower cut-off score value was suitable for Gordonia genus and species identification. Also, the use of discriminatory peaks could aid in the differentiation of G. sputi and G. otitidis from other Gordonia species. Finally, it would be desirable, due to the increase in the description of new pathogenic Gordonia species, to increase the spectra of reference isolates in the MALDI-TOF databases to ensure a correct Gordonia species identification.

Declarations

Authors’ contributions

MEA: writing the first draft. MA: analysis, and interpretation of data by MALDI-TOF MS. PMI: collect the samples, analysis, as well as manuscript review. DV: analysis the samples by molecular methods, as well as manuscript review. JMM: review and discuss the manuscript. All authors read and approved the final manuscript.

Acknowledgements

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

All the data as well as material are disposable at Donostia University Microbiology department’s database as well as in its frozen storage collection.

Consent for publication

Not applicable.

Ethical approval and consent to participate

“All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.”

Funding

Not applicable.

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Authors’ Affiliations

(1)
CIBER de Enfermedades Respiratorias-CIBERES, Madrid, Spain
(2)
Preventive Medicine and Public Health Department, University of Basque Country, Donostia, Spain
(3)
Microbiology Department, Donostia University Hospital-Biodonostia Health Institute, Paseo Dr. Beguiristain s/n, 20014 Donostia-San Sebastian, Spain

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