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. 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 ionizationtime 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...

Evaluation of the Bruker MALDI Biotyper for Identification of Gram-Positive Rods: Development of a Diagnostic Algorithm for the Clinical Laboratory

“…Identification rates for the direct transfer-formic acid method were comparable to those of the ethanol-formic acid extraction procedure that is considered the gold standard and which is used to generate the reference database. These findings are in agreement with previous reports which indicated that on-target treatment with formic acid or ethanol-formic acid extraction improved identification of GPR by MALDI-TOF MS (13,14,24,26). However, the ethanol-formic acid procedure includes various manual preparation steps and is time-consuming (22).…”

“…Identification by MALDI-TOF MS has been analyzed for single genera of GPR, such as Corynebacterium (13)(14)(15), Actinomyces (16), Nocardia (17), Listeria (18), anaerobic GPR (19), and difficult-to-identify GPR (20,21). These studies were mostly done using the Bruker MALDI Biotyper system and indicated that sample preparation by the direct transfer procedure without additional modification is not sufficient for accurate identification of GPR but that a chemical extraction method is required.…”

Bacteriological characteristics of Arcanobacterium haemolyticum isolated from seven patients with skin and soft-tissue infections

“…In addition to the reverse CAMP test, the API Coryne test was used to accurately identify A. haemolyticum, as in previous studies (Soto et al, 1994;Hassan et al, 2009;García-de-la-Fuente et al, 2012). As in previous reports, MALDI-TOF MS was also useful for the rapid identification of A. haemolyticum (Hijazin et al, 2012;Vila et al, 2012). The identification of A. haemolyticum strains at the species level was confirmed by the PCR amplification of pld, which encodes phospholipase D.…”

Clinical Significance of Commensal Gram-Positive Rods Routinely Isolated from Patient Samples

“…Concordant results upon direct Gram staining increase the confidence level for an involvement in infection and are useful for retrospective reviews of data sets, but the decision to select isolates for species-level identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in most laboratories is independent of direct Gram stain results. With the advent and incorporation of MALDI-TOF MS in the clinical microbiology laboratory, the majority of these suspicious diphtheroids can now be routinely identified to the species level (7,8).…”