Identification of Clinically Relevant Corynebacterium spp., Arcanobacterium haemolyticum, and Rhodococcus equi by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

Abstract

The identification of 83 Corynebacterium, 13 Arcanobacterium haemolyticum, and 10 Rhodococcus equi strains by conventional methods (API Coryne complemented with 16S rRNA gene sequence analysis) was compared with matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry identification. The correlation between API and MALDI-TOF results was 89%. MALDI-TOF is a rapid and accurate system for identification of the above-mentioned microorganisms.

TEXT

Corynebacteria are widespread throughout nature. Pathogenic Corynebacterium species include Corynebacterium diphtheriae and nondiphtheroid Corynebacterium. The nondiphtheroid Corynebacterium species are found in the mucosa and normal skin flora of humans and animals. Some species, such as Corynebacterium amycolatum, Corynebacterium jeikeium, Corynebacterium striatum, Corynebacterium urealyticum, and Corynebacterium xerosis, are relevant human pathogens, mainly infecting immunocompromised patients (4). The API Coryne V2.0 system (bioMérieux, Marcy l'Etoile, France), complemented with conventional phenotypic testing, is most commonly used in routine laboratories for identification of these microorganisms. However, this method is time-consuming and does not always give reliable identification at the species level (2). Identification by means of 16S rRNA sequencing is more specific but is slow and expensive.

Arcanobacterium haemolyticum is an obligate parasite of the pharynx of humans; sporadically, it causes pharyngeal or skin lesions (11). Rhodococcus equi is the most important pathogenic species of the genus Rhodococcus, causing several infections such as necrotizing pneumonia and enteritis, mainly in immunocompromised patients such HIV-positive patients (14). A. haemolyticum and R. equi are also routinely identified using the API Coryne V2.0 system. In the case of R. equi, reliable identification frequently requires confirmation by molecular methods, including PCR and DNA sequencing (7).

In recent years, matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) has been increasingly applied in clinical microbial diagnostics for species identification of bacterial and fungal pathogens (13). MALDI-TOF MS identified successfully at the species level a group of 115 Corynebacterium clinical isolates, including 78 C. diphtheriae and 37 nondiphtheroid corynebacterial isolates (6). In a recent study (3), MALDI-TOF discriminated Corynebacterium aurimucosum from Corynebacterium minutissimum, two closely related Corynebacterium species previously considered difficult to differentiate (5).

The aim of this study was to determine whether MALDI-TOF MS can be used as a routine method for fast and reliable identification of Corynebacterium clinical isolates at the species level in our laboratories. Eighty-three clinical strains isolated at the Clinical Microbiology Laboratory, Hospital Universitario Marqués de Valdecilla, Santander, Spain, were initially identified by API Coryne V2.0 and other conventional phenotypic methods (4) as C. jeikeium (18 strains), Corynebacterium pseudodiphthericum (16 strains), C. striatum (15 strains), C. amycolatum (12 strains), C. urealyticum (9 strains), Corynebacterium glucuronolyticum (3 strains), Rhodococcus equi (11 strains), and Arcanobacterium haemolyticum (13 strains). The strains C. striatum ATCC 6940 and C. urealyticum ATCC 43042 were also included in the study. Toxin-producing species such as Corynebacterium diphtheriae were not examined in this study because of a lack of samples in this particular collection. The strains were analyzed by MALDI-TOF MS through the following procedure: a small amount of a colony grown on blood agar was transferred to a metallic MALDI-TOF MSP 96 plate (Bruker Daltonik GmbH, Bremen, Germany) and thereafter covered with 1 μl of matrix (saturated alpha-cian-4-hydroxycinamic acid–50% acetonitrile–2.5% trifluoroacetic acid). The plate was then left to dry at room temperature for 5 min. When that first attempt at identification by MS did not show results with a high level of confidence, samples were identified using formic acid-acetonitrile extraction. The reference strain Escherichia coli K-12 (genotype GM48) was used as a standard for calibration and as a reference for quality control. Measurements were performed using a Microflex II mass spectrometer (Bruker Daltonik) equipped with a 60-Hz laser. The proteic spectra obtained with the spectrometer were processed with Bruker MALDI Biotyper v2.0 software (Bruker Daltonik GmbH) and compared with the reference spectra present in the database, showing the 10 most similar patterns for each isolate. The similarity of patterns is demonstrated as a score (≥2, identification at species level; 1.7 to 1.999, identification at genus level; <1.7, no reliable identification). Discrepancies between API Coryne V2.0 and MALDI-TOF MS identification were resolved by partial sequencing of the 16S rRNA gene and sequencing of the rpoB gene (1).

All the isolates identified as C. amycolatum, C. glucuronolyticum, C. striatum, C. urealyticum, and A. haemolyticum by API Coryne V2.0 were also identified by MALDI-TOF MS as such, scoring ≥2 (Table 1). Of 19 strains identified as C. jeikeium by API Coryne V2.0 testing, 1 was identified as C. amycolatum by MALDI-TOF MS. 16S rRNA sequencing assigned this isolate to C. amycolatum. C. jeikeium is recognized as one of the Corynebacterium species most frequently associated with human infectious diseases (8), causing sepsis, endocarditis, pneumonia, and other infections, while C. amycolatum is considered an emergent pathogen (9). Of 7 strains identified as C. minutissimum by API Coryne V2.0, 3 were also identified by MALDI-TOF MS, 3 were assigned to C. aurimucosum, and 1 was assigned to C. amycolatum (Table 1). 16S rRNA sequencing confirmed that the isolates did indeed belong to the species identified by MALDI-TOF. However, when the rpoB gene was sequenced, the results obtained by MALDI-TOF were confirmed, with the exception of two C. minutissimum strains, which were identified as C. aurimucosum; therefore, further investigation is needed to evaluate the use of MALDI-TOF MS to identify this species. It has been previously reported that, because their biochemical profiles are very similar, API Coryne V2.0 did not discriminate among these three species, needing complementary tests for a reliable identification (12). Discrepancies between API Coryne V2.0 and MALDI-TOF MS results were also found with 5 of the 16 strains identified as C. pseudodiphthericum by API Coryne V2.0, all 5 of which were assigned to C. propinquum by MALDI-TOF MS. 16S rRNA sequencing confirmed that the 5 isolates were C. propinquum. Indeed, there was also total correlation between the identification by MALDI-TOF MS and sequencing of the rpoB gene. C. pseudodiphthericum and C. propinquum are part of the oropharyngeal microbiota. They have been reported to be among the species causing pneumonia and endocarditis. The two species are phenotypically analogous, but most C. pseudodiphthericum strains produce urease, whereas C. propinquum strains are urease negative.

Table 1.

Comparative results of the identification methods used in this study

Microorganism	Total no. of strains	No. of strains identified by indicated method
		API Coryne V2.0	MALDI-TOF	16S rRNA sequencing
Corynebacterium amycolatum	12	12	12
Corynebacterium glucuronolyticum	3	3	3
Corynebacterium jeikeium	19	19	18 C. jeikeium
			1 C. amycolatum	1 C. amycolatum
Corynebacterium minutissimum	7	7	3 C. minutissimuma
			3 C. aurimucosum	3 C. aurimucosum
			1 C. amycolatum	1 C. amycolatum
Corynebacterium pseudodiphtheriticum	16	16	11 C. pseudodiphtheriticumb
			5 C. propinquuma	5 C. propinquum
Corynebacterium striatum	16	16	16
Corynebacterium urealyticum	10	10	10
Rhodococcus equi	11	11	10 R. equi
			1 Dietzia maris	1 D. maris
Arcanobacterium haemolyticum	13	13	13

^aTwo of these three strains were identified as C. aurimucosum by sequencing the rpoB gene.

^bAll C. pseudodiphteriticum and C. propinquum isolates identified by MALDI-TOF MS were confirmed by sequencing the rpoB gene.

Of 11 isolates presumptively identified as R. equi by API Coryne V2.0, MALDI-TOF MS identified all except 1, which was identied as Dietzia maris. 16S rRNA gene sequencing identified this isolate as D. maris. We have recently reported that 8 of 15 clinical isolates presumptively identified by API Coryne as R. equi at Hospital Marqués de Valdecilla, Santander, Spain, belonged in fact to the genus Dietzia (10).

Since most of the nondiphtheroid strains of Corynebacterium, A. haemolyticum, and R. equi are actually well-recognized human pathogens, their reliable identification at the species level is necessary. API Coryne V2.0 is, at this time, the method most commonly used for identification of these bacteria in routine microbiological laboratories, but its trustworthiness at the species level is limited. Moreover, it requires at least 16 h for processing after isolation of suspicious colonies from screening plates. In contrast, MALDI-TOF MS analysis is much faster, rendering species identification of one isolate in less than 10 min. This study demonstrates that MALDI-TOF MS is a rapid and consistent system for routine identification at the species level of clinical isolates of the above-mentioned microorganisms.