Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Abstract

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n = 180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n = 156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Introduction

Bovine mastitis is the most frequent and costly disease in the dairy industry and affects both the quality and yield of milk (Halasa et al., 2007). Corynebacterium spp. have been identified as common agents of subclinical intramammary infections (IMI) and are thought to account for 10–25% of the reported cases of this infection (Haltia et al., 2006, Schukken et al., 2009). These mastitis pathogens are generally associated with moderate increases in the somatic cell counts (SCC) from the affected dairy cows (Schukken et al., 2009).

Despite the high percentage of mastitis infections caused by Corynebacterium spp., these organisms cannot be accurately identified at the species level using conventional bacteriological techniques. Accordingly, knowledge regarding the number of species is limited, and the role of Corynebacterium in bovine mastitis is poorly understood. Moreover, conventional tests to identify Corynebacterium spp. in bovine milk have a reported misidentification rate of nearly 30% (Coyle and Lipsky, 1990, Watts et al., 2000) and, in most cases, isolate identification is used to distinguish the isolates from other more pathogenic microorganisms such as Nocardia spp. (Hogan et al., 1999). Hence, simple and rapid methods could assist in the identification of Corynebacterium spp. isolated from milk samples and allow a better understanding of their role in mastitis.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) can be used to rapidly detect and characterize bacterial strains; this method can detect a large range of biomolecules within complex mixtures at a high speed and sensitivity level (Maier et al., 2006, Welker, 2011). Our group has previously reported the use of MALDI-TOF MS to identify bacteria isolated from subclinical bovine mastitis samples (Barreiro et al., 2010, Braga et al., 2013); however these studies did not focus on the identification of mastitis-causing Corynebacterium spp. In bacterial characterization, the major use of MS is the direct acquisition of a unique protein biomarker profile for each microorganism from an intact sample (Welker, 2011).

Corynebacterium spp. isolated from clinical human samples have been identified by MS (Theel et al., 2012) but the identification of mastitis-causing Corynebacterium spp. has not yet been reported. Therefore, this study aimed to evaluate the ability of MALDI-TOF MS to identify Corynebacterium spp. isolated from the milk samples of dairy cows with IMI.