Research Article - (2011) Volume 0, Issue 0
Corynebacterium striatum is frequently encountered in the routine clinical microbiology laboratory. It is widely disseminated in the environment and constitutes part of the normal micro-biota of the skin and mucous membrane. Identification of this species by biochemical methods remains difficult and several misidentifications have been reported previously. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for the identification of this microorganism was designed based on the hypervariable region of the polymorphic RNA polymerase β-subunit gene (rpoB). All available Corynebacterium rpoB sequences were analyzed by computerassisted restriction analysis. The rpoB PCR-RFLP pattern predicted by using endonucleases MseI and NlaIV clearly differentiated C. striatum from all other Corynebacterium species. This method was successfully applied for the reliable identification of 67 C. striatum clinical isolates and can be used for the timely detection of infected patients or for epidemiological studies.
The genus Corynebacterium is composed of Gram positive bacteria, facultative anaerobe, that are widely distributed . 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 . Several molecular methods have been used to identify Corynebacterium species including DNA-DNA hybridization , sequence analyses of 16S rRNA and rpoB genes  and rpoB gene RFLP . 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 . Pavan et al.  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).
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 . Enzyme restriction patterns for the rpoB amplified region of each Corynebacterium species were generated using REBASE program . 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.
|Species and rpoB Genbank Acession No||Predicted sizes of fragments (bp) after amplicon digestion with:|
|Mse I||Nla IV|
|C. striatum AY492267||323-123||138-308|
|C. simulans AY492264||nr||138-308|
|C. ulcerans AY492271||nr||nr|
|C. amycolatum AY492241||nr||nr|
|C. minutissimum AY492235||nr||369-77|
|C. maginleyi AY492276||98-348||278-91-77|
|C. pseudotuberculosis AY492239||351-95||nr|
|C. glucuronolyticum AY492256, C. seminale AY492263||311-123||nr|
|C. imitans AY492259||nr||nr|
No restriction sites for these enzymes were found in the rpoB sequences of the following Corynebacterium species: C. accolens AY492242, C. ammoniagenes AY492243, C. argentoratense AY492249, C. aurimucosum AY492282, C. auris AY492234, C. auriscanis AY492244, C. bovis AY492236, C. camporealensis AY492246, C. capitovis AY492247, C. casei EU616817, C. confusum AY492248, C. coyleae AY492250, C. diphtheriae AY492230, C. durum AY492252, C. efficiens AP005215, C. falsenii AY492253, C. felinum AY492254, C. flavescens AY492255, C. freneyi AY492237, C. genitalium EU616818, C. jeikeium AY492231, C. lipophiloflavum AY492260, C. mastitidis AY492281, C. matruchotii AY492238, C. mucifaciens AY492261, C. mycetoides AY492262, C. phocae AY492277, C. propinquum AY492279, C. pseudodiphtheriticum AY492232, C. pseudogenitalium AY581868, C. pyruviciproducens FJ899747, C. riegelii AY492278, C. singulare AY492280, C. spheniscorum AY492283, C. sundsvallense AY492268, C. terpenotabidum AY492269, C. testudinoris AY492284, C. thomssenii AY492270, C. tuberculostearicum AY581869, C. urealyticum AY492275, C. ureicelerivorans FJ392022/ FJ392020/ FJ392018/ FJ392029/FJ392021, C. variabile AY492272, C. xerosis AY492233
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).
To confirm the identification, all strains were analyzed by MALDI TOF MS (Bruker Daltonics, GmbH) as previously described . 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 . 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 . 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.
|Strains identified by n||Final Identification|
|Api Coryne Strips (number of strains n)||PCR-RFLP-rpoB gene Profil|
|C. striatum/amycolatum (n= 67)||323-123||138-308||67C. striatum|
|C. striatum/ amycolatum (n=18)||NR||NR||14 C. amycolatum 2 C. aurimucosum 1 C. singulare 1C. imitans|
|C. macginleyi (n=1)||98-348||278-91-77||1 C. macginleyi|
|C. diphtheriae (n=1)||NR||NR||1 C. diphtheriae|
|C. jeikeuim (n=1)||NR||NR||1 C. jeikeuim|
|C. coylae/ afermentans (n=1)||NR||NR||1 C. coylae|
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.