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Title: A novel method for simultaneous Enterococcus species identification/typing and van genotyping by high resolution melt analysis.
Austin Authors: Gurtler, Volker;Grando, Danilla;Mayall, Barrie C;Wang, Jenny;Ghaly-Derias, Shahbano
Affiliation: Department of Pathology, Austin Hospital, Heidelberg 3084, Australia
Issue Date: 14-May-2012
Publication information: Journal of Microbiological Methods 2012; 90(3): 167-81
Abstract: In order to develop a typing and identification method for van gene containing Enterococcus faecium, two multiplex PCR reactions were developed for use in HRM-PCR (High Resolution Melt-PCR): (i) vanA, vanB, vanC, vanC23 to detect van genes from different Enterococcus species; (ii) ISR (intergenic spacer region between the 16S and 23S rRNA genes) to detect all Enterococcus species and obtain species and isolate specific HRM curves. To test and validate the method three groups of isolates were tested: (i) 1672 Enterococcus species isolates from January 2009 to December 2009; (ii) 71 isolates previously identified and typed by PFGE (pulsed-field gel electrophoresis) and MLST (multi-locus sequence typing); and (iii) 18 of the isolates from (i) for which ISR sequencing was done. As well as successfully identifying 2 common genotypes by HRM from the Austin Hospital clinical isolates, this study analysed the sequences of all the vanB genes deposited in GenBank and developed a numerical classification scheme for the standardised naming of these vanB genotypes. The identification of Enterococcus faecalis from E. faecium was reliable and stable using ISR PCR. The typing of E. faecium by ISR PCR: (i) detected two variable peaks corresponding to different copy numbers of insertion sequences I and II corresponding to peak I and II respectively; (ii) produced 7 melt profiles for E. faecium with variable copy numbers of sequences I and II; (iii) demonstrated stability and instability of peak heights with equal frequency within the patient sample (36.4±4.5 days and 38.6±5.8 days respectively for 192 patients); (iv) detected ISR-HRM types with as much discrimination as PFGE and more than MLST; and (v) detected ISR-HRM types that differentiated some isolates that were identical by PFGE and MLST. In conjunction with the rapid and accurate van genotyping method described here, this ISR-HRM typing and identification method can be used as a stable identification and typing method with predictable instability based on recombination and concerted evolution of the rrn operon that will complement existing typing methods.
Gov't Doc #: 22658426
DOI: 10.1016/j.mimet.2012.05.002
Type: Journal Article
Subjects: Bacterial Proteins.genetics
Base Sequence
Consensus Sequence
DNA, Bacterial.genetics
DNA, Ribosomal Spacer.genetics
Enterococcus faecalis.classification.genetics.isolation & purification
Enterococcus faecium.classification.genetics.isolation & purification
Genes, Bacterial
Genotyping Techniques
Gram-Positive Bacterial Infections.microbiology
INDEL Mutation
Molecular Sequence Data
Multilocus Sequence Typing
Peptide Synthases.genetics
Principal Component Analysis
Transition Temperature
Vancomycin Resistance.genetics
Appears in Collections:Journal articles

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