Modified 16S–23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
Fast and reliable identification of bacteria to at least the species level is currently the basis for correct diagnosis and appropriate treatment of infections. This is particularly important in the case of bacteria of the genus Enterococcus, whose resistance profile is often correlated with their species (e.g. resistance to vancomycin). In this study, we evaluated restriction endonuclease analysis of the 16S–23S rRNA gene intergenic transcribed spacer (ITS) region for species identification of Enterococcus. The utility of the method was compared with that of phenotypic methods [biochemical profile evaluation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS)]. Identification was based on 21 Enterococcus reference strains, of the species E. faecalis, E. faecium, E. hirae, E. durans, E. casseliflavus, E. gallinarum, E. avium, E. cecorum and E. columbae, and 47 Enterococcus field strains isolated from pigs. Restriction endonuclease analysis of the ITS-PCR product using HinfI, RsaI and MboI, in the order specified, enabled species differentiation of the Enterococcus reference and field strains, and in the case of the latter, the results of species identification were identical (47/47) to those obtained by MALDI-TOF MS. Moreover, as a result of digestion with MboI, a unique restriction profile was also obtained for the strains (3/3) identified by MALDI-TOF MS as E. thailandicus. In our opinion, restriction endonuclease analysis of the 16S–23S rRNA gene ITS region of Enterococcus may be a simple and relatively fast (less than 4 h) alternative method for identifying the species occurring most frequently in humans and animals.
AriasC. A., RobredoB., SinghK. V., TorresC., PanessoD., MurrayB. E.2006; Rapid identification of Enterococcus hirae and Enterococcus durans by PCR and detection of a homologue of the E. hirae mur-2 gene in E. durans. J Clin Microbiol 44:1567–1570 [View Article][PubMed]
ChampagneJ., DiarraM. S., RempelH., ToppE., GreerC. W., HarelJ., MassonL.2011; Development of a DNA microarray for enterococcal species, virulence, and antibiotic resistance gene determinations among isolates from poultry. Appl Environ Microbiol 77:2625–2633 [View Article][PubMed]
DevrieseL. A., PotB., CollinsM. D.1993; Phenotypic identification of the genus Enterococcus and differentiation of phylogenetically distinct enterococcal species and species groups. J Appl Bacteriol 75:399–408 [View Article][PubMed]
DomigK. J., MayerH. K., KneifelW.2003; Methods used for the isolation, enumeration, characterisation and identification of Enterococcus spp. 2. Pheno- and genotypic criteria. Int J Food Microbiol 88:165–188 [View Article][PubMed]
Dutka-MalenS., EversS., CourvalinP.1995; Detection of glycopeptide resistance genotypes and identification to the species level of clinically relevant enterococci by PCR. J Clin Microbiol 33:24–27[PubMed]
HarwoodV. J., DelahoyaN. C., UlrichR. M., KramerM. F., WhitlockJ. E., GareyJ. R., LimD. V.2004; Molecular confirmation of Enterococcus faecalis and E. faecium from clinical, faecal and environmental sources. Lett Appl Microbiol 38:476–482 [View Article][PubMed]
HayesJ. R., EnglishL. L., CarterP. J., ProescholdtT., LeeK. Y., WagnerD. D., WhiteD. G.2003; Prevalence and antimicrobial resistance of Enterococcus species isolated from retail meats. Appl Environ Microbiol 69:7153–7160 [View Article][PubMed]
JacksonC. R., Fedorka-CrayP. J., BarrettJ. B.2004; Use of a genus- and species-specific multiplex PCR for identification of enterococci. J Clin Microbiol 42:3558–3565 [View Article][PubMed]
JensenM. A., WebsterJ. A., StrausN.1993; Rapid identification of bacteria on the basis of polymerase chain reaction-amplified ribosomal DNA spacer polymorphisms. Appl Environ Microbiol 59:945–952[PubMed]
JurkovičD., KrizkováL., DusinskýR., BelicováA., SojkaM., KrajčovičJ., EbringerL.2006; Identification and characterization of enterococci from bryndza cheese. Lett Appl Microbiol 42:553–559[PubMed]
KnijffE., DellaglioF., LombardiA., AndrighettoC., TorrianiS.2001; Rapid identification of Enterococcus durans and Enterococcus hirae by PCR with primers targeted to the ddl genes. J Microbiol Methods 47:35–40 [View Article][PubMed]
KühnI., IversenA., BurmanL. G., Olsson-LiljequistB., FranklinA., FinnM., AarestrupF., SeyfarthA.-M., BlanchA. R. et al.2000; Epidemiology and ecology of enterococci, with special reference to antibiotic resistant strains, in animals, humans and the environment. Example of an ongoing project within the European research programme. Int J Antimicrob Agents 14:337–342 [View Article][PubMed]
LavováM., BezekováJ., ČanigováM., KročkoM., DomigK. J.2014; Species identification of enterococci by biochemical test and molecular-genetic methods. Potravinarstvo 8:124–129 [View Article]
LleòM. M., BonatoB., BenedettiD., CanepariP.2005; Survival of enterococcal species in aquatic environments. FEMS Microbiol Ecol 54:189–196 [View Article][PubMed]
MooreD. F., ZhowandaiM. H., FergusonD. M., McGeeC., MottJ. B., StewartJ. C.2006; Comparison of 16S rRNA sequencing with conventional and commercial phenotypic techniques for identification of enterococci from the marine environment. J Appl Microbiol 100:1272–1281 [View Article][PubMed]
NaserS. M., ThompsonF. L., HosteB., GeversD., DawyndtP., VancanneytM., SwingsJ.2005; Application of multilocus sequence analysis (MLSA) for rapid identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151:2141–2150 [View Article][PubMed]
PoyartC., QuesnesG., Trieu-CuotP.2000; Sequencing the gene encoding manganese-dependent superoxide dismutase for rapid species identification of enterococci. J Clin Microbiol 38:415–418[PubMed]
PrakashV. P., RaoS. R., ParijaS. C.2005; Emergence of unusual species of enterococci causing infections, South India. BMC Infect Dis 5:14 [View Article][PubMed]
ReidK. C., CockerillF. R.III, PatelR.2001; Clinical and epidemiological features of Enterococcus casseliflavus/flavescens and Enterococcus gallinarum bacteremia: a report of 20 cases. Clin Infect Dis 32:1540–1546 [View Article][PubMed]
ShewmakerP. L., SteigerwaltA. G., NicholsonA. C., CarvalhoM. G., FacklamR. R., WhitneyA. M., TeixeiraL. M.2011; Reevaluation of the taxonomic status of recently described species of Enterococcus: evidence that E. thailandicus is a senior subjective synonym of “E. sanguinicola” and confirmation of E. caccae as a species distinct from E. silesiacus. J Clin Microbiol 49:2676–2679 [View Article][PubMed]
Modified 16S–23S rRNA intergenic region restriction endonuclease analysis for species identification of Enterococcus strains isolated from pigs, compared with identification using classical methods and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry