Automated ribotyping of vancomycin-resistant Enterococcus faecium isolates

Abstract

Vancomycin-resistant Enterococcus faecium (VREF) strains represent an important threat in hospital infections in the United States and are found at high frequencies in both the community and farm animals in Europe. We evaluated automated ribotyping for interlaboratory reproducibility by using the restriction enzymes EcoRI and BamHI and compared ribotyping to both amplification of fragment length polymorphism (AFLP) analysis and multilocus sequence typing (MLST) to assess its discriminatory power and capacity for the identification of epidemiologically important strains. Of 19 (EcoRI) and 16 (BamHI) isolates tested in duplicate in two laboratories, 18 (95%) and 16 (100%), respectively, showed reproducible ribotypes. These high reproducibility rates were obtained only after manual refinement of the automated fingerprint analysis. A group of 49 VREF strains initially selected to represent 32 distinct AFLP types were separated into 28 EcoRI ribotypes, 25 BamHI ribotypes, and 28 sequence types. Ribotyping with EcoRI and BamHI was able to discern the host-specific genogroups recently disclosed by AFLP typing and MLST and to distinguish most strains containing the esp gene, a marker specific for strains causing hospital outbreaks. An expandable ribotype identification library was created. We recommend EcoRI as the enzyme of choice for automated ribotyping of VREF strains. Given the high level of discrimination of VREF strains, the high rate of interlaboratory reproducibility, and the potential for the identification of epidemiologically important genotypes, automated ribotyping appears to be a very valuable approach for characterizing VREF strains.

FIG.1.

Interlaboratory comparison of automated ribotyping patterns obtained by using EcoRI and BamHI restriction enzymes. The molecular size scale above the patterns is in kilobases. The letter “u” after a strain name corresponds to the second test of the strain (performed in Utrecht with EcoRI and in Copenhagen with BamHI). The reproducibility of ribotyping with BamHI was not tested for strains SB440, SB441, and SB442.The pairs of patterns that were not reproducible after automated categorization but that were reproducible after manual categorization are indicated with arrowheads. The only pair of patterns that was also not reproducible after manual categorization is indicated with diamonds. The arrow indicates the pattern into which a molecular size marker band of 1 kb was incorporated by the automated analysis of the RiboPrinter due to suboptimal electrophoretic migration.

FIG.2.

Overview of the patterns obtained after automated ribotyping with BamHI and EcoRI restriction enzymes of 49 E. faecium strains. Clustering was obtained by using the UPGMA algorithm based on the Dice coefficient calculated from the BamHI patterns. Epidemiologically related strains are indicated by underlining and bold type. SB440, SB441, and SB442 originated from hospital outbreak US-1, and SB401, SB437, and SB438 were from outbreak UK-1 (28). Strains SB448 and SB449 originated from a hospital outbreak in France. Strains SB407 and SB409 were derived from the same patient. The isolation sources of the isolates are indicated by the following abbreviations: P, poultry; PF, poultry farmer; HP, hospitalized patient; D, dog; C, cat; S, swine; VC, veal calf; HV, human volunteer. Strain names, EcoRI and BamHI ribogroups, sources, AFLP types, AFLP genogroups, MLST types, and presence or absence of the esp gene are indicated in the respective columns. The DNA molecular size scale is given above the fingerprint patterns.