The genus Corynebacterium is composed of Gram positive bacteria, facultative anaerobe, that are widely distributed [1]. The identification of Corynebacterium species is difficult because it always needs particular techniques or a big number of biochemical tests that are not available in API system [2]. Several molecular methods have been used to identify Corynebacterium species including DNA-DNA hybridization [3], sequence analyses of 16S rRNA and rpoB genes [4] and rpoB gene RFLP [1]. However, the 16S rRNA genes sequence analysis, which is the most used to identify bacteria or to determine their phylogenetic relationships, has limits in the identification of Corynebacterium species because of his low intragenus polymorphism. The rpoB gene is polymorphic enough to be used for the accurate identification of Corynebacterium species [5]. Pavan et al. [1] demonstrated that rpoB RFLP analysis can be used for the reliable identification of C. pseudotuberculosis strains isolated from sheep. In our study, we investigated the application of PCR-RFLP analysis of the hypervariable sequence of rpoB gene for the speciation of Corynebacterium striatum strains (Figure 1).

Figure 1: Schematic representation of the rpoB gene showing the restriction sites of the endonucleases for C. striatum .

Bacterial strains

Eighty five strains identified as C. striatum/amycolatum by the routine assays and Api Coryne V.2 strips were studied. Four others clinically relevant Corynebacterium spp: C. macginleyi, C. diphtheriae, C. coylae and C. jeikeuim were included in this study. The strains were collected from multiple clinical sources, including blood, tissue, urine, wound, respiratory specimens and others sources, during a period of five years (2007-2013) in the Universitary Hospital F. Hached, Tunisia. C. striatum ATCC6940 was used as control.

Choice of restriction enzymes

Initially all the rpoB hypervariable sequences publicly available in GenBank database belonging to 62 Corynebacterium species were aligned [1]. Enzyme restriction patterns for the rpoB amplified region of each Corynebacterium species were generated using REBASE program [6]. The predicted MseI and NlaIV restriction fragments of the rpoB amplicon in Corynebacterium species using REBASE program are listed in Table 1. The majority of Corynebacterium species did not contain a restriction site for these endonucleases.

Table 1: Predicted MseI and NlaIV restriction fragments of the rpoB amplicon in different Corynebacterium species using the REBASE program.

rpoB PCR-RFLP analysis

All strains were cultured overnight, on 5% horse blood agar in 5% CO₂ at 37°C. The DNA from each strain was extracted by QIAamp DNA MiniKit (Qiagen GmbH, Germany), following the manufacturer’s instructions. PCR was carried out in a final volume of 50 µl as described previously using oligonucleotides C2700F and C3130R [1,5]. Amplified products were separated in agarose gel 2% and were visualized by ethidium bromide staining. Following the PCR, 12 µl of amplified products were digested using endonucleases MseI and NlaIV in two separate reactions according to manufacturer’s guidelines. RFLP products were analyzed using 2% agarose gel, at 100V for 1 hour. A 100-pb molecular weight marker was used as a molecular size standard (Figure 2).

Figure 2: Identification of Corynebacterium striatum strains by rpoB -PCR-RFLP. (a) Lane 1 and 6: 100 bp ladders. Lane 2-5: rpoB gene MseI restriction from C. striatum . Lane 7-11: rpoB gene MseI restriction from C. amycolatum , C. singulare , C. aurimucosum , C. simulans and C. imitans respectively. Lane 12: Negative control (5 µl of water instead of DNA). (b) Lane 1: 100 bp ladder. Lane 2: Negative control. Lane 3-4: rpoB gene NlaIV restriction from C. imitans (n=1) and C. striatum (n=2).

MALDI-TOF-MS analyses

To confirm the identification, all strains were analyzed by MALDI TOF MS (Bruker Daltonics, GmbH) as previously described [7]. Briefly, a portion of a colony was smeared onto a 96-well target plate, and after drying, it was covered using 1 μl of α-cyano-4-hydroxycinnamic acid (CHCA) matrix solution. When it was dry, the target plate was loaded into the machine, which was equipped with a 337-nm nitrogen laser. The spectra were analyzed using the Biotyper 2.0 software (Bruker, Karlsruhe, Germany). The identification criteria were chosen according to the cutoffs proposed by the manufacturers. Identifications with scores above 2 and between 1.7 and 2 were considered to be reliable at the species and genus levels, respectively. Identification scores below 1.7 were considered unacceptable. When the MALDI-TOF-MS identification was inconclusive, 16S rRNA gene sequencing was performed.

As a result of their being normal human microbiota, Corynebacterium species are commonly considered as contaminants. Because of this and of challenges in identification, they have not received a great deal of attention [8]. Identification of putative pathogenic Corynebacterium is crucial. So far, this has been done biochemically, with Api Coryne strips which takes at least 16 hours after isolation of suspicious colonies from screening plates (typically small grayish colonies, mostly translucent, positive catalase reaction and Gram positive coryneform rods in the form of Chinese letters), and may often yielded unreliable or ambiguous results [7,8]. In this study, we distinguish 2 different colonies’ morphology. Seventy one isolates produced on Columbia agar base with 5% horse blood non-hemolytic, creamy white to yellowish with an entire edge colonies. However, for 15 isolates, colonies were flat, dry, whitish-gray and matte. Using biochemical tests, these strains were identified as C. striatum/amycolatum. In Api Coryne database C. amycolatum, C. minutissimum, and C. striatum gave the same code: (2-3)100(1-3)(0-2)(4-5). Their differential identification by biochemical tests remains difficult, and several misidentifications have been previously reported [9-11]. Although they are genetically different, these species share many phenotypic characteristics and we need supplementary tests to differentiate them [12]. The RFLP analysis of the amplified sequence of Corynebacterium strains identified as C. striatum/ amycolatum by Api Coryne strips indicated that 67 strains presented exactly the same MseI and NlaVI restriction fragments corresponding to predicted patterns for C. striatum. By MALDI-TOF-MS identification, these strains were assigned to C. striatum with scores >2.000. However, 18 strains identified as C. striatum/amycolatum by Api Coryne did not contain restriction sites for MseI and NlaIV. These strains were identified as C. amycolatum (n=14), C. aurimucosum (n=2), C. imitans (n=1) and C. singulare (n=1). The assay was also successfully applied to differentiate C. striatum from other clinically relevant Corynebacterium spp. including C. macginleyi, C. diphtheriae, C. jeikeuim and C. coylae/afermentans (Table 2). Furthermore, the technique proposed has the potential to differentiate C. striatum from the other biochemically and genetically related Corynebacterium spp: C. amycolatum, C. minutissimum, C. simulans, C. ulcerans and C. aurimucosum, none of which have the same restriction sites for NlaIV or MseI in the rpoB region analyzed. It must be taken into account that several base changes would be required in order to change the restriction sites so that a strain from other Corynebacterium species acquired the pattern of C. striatum.

Table 2: Comparison of the results obtained using different methods of identification.

The present study proposes a molecular method involving the PCR-mediated amplification of an internal rpoB region followed by RFLP-analysis. This method has been designed for the identification of C. striatum, distinguishing it from other Corynebacterium species, including all members of this genus of importance in clinical medicine, and from genetically related pathogens such as C. simulans and C. ulcerans. The PCR-RFLP technique described in this work has been experimentally tested to differentiate C. striatum from C. amycolatum and C. minutissimum that produces similar Api Coryne codes. This assay provides a rapider diagnostic tool than biochemical assays or 16S RNA sequencing, for the identification of clinical C. striatum strains and for the discrimination between this species and other related pathogenic bacteria.

We acknowledge Pr. René Courcol from Centre Hospitalier Universitaire de Lille, Institut de Microbiologie F-59037 Lille Cedex, France for the MALDI-TOF-MS analysis.