title: 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.
abstract: 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.
authors.affiliation: University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.
authors.forename: Aneta
authors.initials: A
authors.lastname: Nowakiewicz
authors.affiliation: University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.
authors.forename: Grażyna
authors.initials: G
authors.lastname: Ziółkowska
authors.affiliation: State Veterinary Laboratory, Droga Męczenników Majdanka 50, 20-325 Lublin, Poland.
authors.forename: Przemysław
authors.initials: P
authors.lastname: Zięba
authors.affiliation: University of Life Sciences, Faculty of Veterinary Medicine, Institute of Biological Bases of Animal Diseases, Sub-Department of Veterinary Microbiology, Akademicka 12, 20-033 Lublin, Poland.
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Trościańczyk
authors.affiliation: University of Life Sciences, Faculty of Veterinary Medicine, Department of Epizootiology and Clinic of Infectious Diseases, Głęboka 30, 20-612 Lublin, Poland.
authors.forename: Tomasz
authors.initials: T
authors.lastname: Banach
authors.affiliation: University of Life Sciences, Faculty of Veterinary Medicine, Department of Preclinical Veterinary Sciences, Sub-Department of Pharmacology, Akademicka 12, 20-033 Lublin, Poland.
authors.forename: Cezary
authors.initials: C
authors.lastname: Kowalski
mesh_terms: D000818:Animals; D015373:Bacterial Typing Techniques / Q000662:veterinary*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D016983:Enterococcus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology / Q000662:veterinary; D016133:Polymerase Chain Reaction / Q000662:veterinary*; D012150:Polymorphism, Restriction Fragment Length; D015183:Restriction Mapping / Q000662:veterinary; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000662:veterinary; D013552:Swine; D013553:Swine Diseases / Q000382:microbiology; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer