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-offlight 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.

Characterization of Multidrug Resistant E. faecalis Strains from Pigs of Local Origin by ADSRRS-Fingerprinting and MALDI -TOF MS; Evaluation of the Compatibility of Methods Employed for Multidrug Resistance Analysis

“…The gradual development of the proteomic technique MALDI-TOF MS has provided new opportunities for precise microbiological analysis. Numerous studies have shown that MALDI-TOF MS is a rapid, reliable, and cost-effective technique for identification of different groups of microorganisms [ 20 , 58 – 61 ], including Enterococcus [ 25 , 62 , 63 ]. In our study, we also obtained high log (score) values (> 2) for all the strains tested and the results were consistent with those of molecular identification.…”

“…Identification to the genus Enterococcus was performed as previously described [ 12 ]. Species identification was performed using 16S–23S rRNA intergenic region restriction endonuclease analysis according to previously described protocols [ 25 ].…”

Correlation between Infective Factors and Antibiotic Resistance inEnterococciClinical Isolates in West of Iran

“…In this study, there was a slight discrepancy between biochemical and PCR results for species identification, which is in accordance with other studies. 20 21 The main reason for this discrepancy is the similarities between Enterococcus species and the high phenotypic variation within individual species. PCR is a more accurate technique in comparison to the biochemical approach, so PCR results were preferred for the strains with discrepant identification.…”

Prevalence and antibiotic resistance of Enterococcus strains isolated from poultry

“…A relatively new identification method using a MALDI-TOF MS system with prior formic acid extraction has provided excellent diagnostic results and reduced identification time (Seng et al, 2009;Wieser et al, 2012). MALDI-TOF MS is a rapid and accurate technique for the identification of various types of Grampositive or Gram-negative bacteria (Kosikowska et al, 2014;Marek et al, 2015;Nowakiewicz et al, 2015).…”