Identification methods of enterococci

Typing methods

Systems for typing of microorganisms can be divided into genotypic and phenotypic:

Genotypic methods

Molecular typing of enterococci in outbreak situations is commonly performed by pulsed-field gel electrophoresis (PFGE), including preparation of chromosomal DNA, cleavage with restriction enzymes and PFGE have been the methods of choice when investigating clonal relationships among enterococci. Banding patterns produced by each organism are matched, and this information is combined with epidemiologic data to determine relatedness between strains. The guidelines for interpretation of relatedness and clonality (<7 band differences between strains) as proposed by Fred Tenover have been considered the golden standard in such investigations. PFGE offers high reproducibility and has a high discriminatory power which is useful when investigating local outbreaks during shorter time periods (e.g. six months) but can be a disadvantage when investigating clonal relationships over longer time periods. One single deletion or insertion of a base pair in the genome of the bacteria can result in three band differences and insertion of a transposon in vitro more than 6 band differences in the same strain. However, investigations of VRE strains in long-term colonized patients over periods up to 160 days have shown strains to be genetically stable, suggesting that large changes in the genome occur infrequently among clinical isolates in nature. Another drawback of PFGE is that it is a labor-intensive method and other methods may be more suitable when typing large numbers of isolates. Other molecular methods, such as contour-clamped homogeneous electric field electrophoresis patterns, amplified ribosomal DNA spacer polymorphisms, and randomly amplified polymorphic DNA analysis, have been used to identify enterococci at the species level. However, it is difficult to adapt these tests for use in clinical microbiology laboratories because of their complexity. An Enterococcus spp. assay based on the hybridization of rRNA genes is commercially available for culture confirmation (6). The sensitivity of this assay is unsatisfactory for direct detection from clinical specimens.

To further overcome the shortcomings of PFGE, even more sophisticated genetic typing systems such as amplified fragment length polymorphism (AFLP) and multilocus sequence typing (MLST) have been developed.

These are even more labor intensive and expensive methods that include PCR of genomic restriction fragments (AFLP) or direct sequencing of defined sections of house keeping genes of the bacteria MLST. A power computer then constructs dendrograms based on the PCR and sequence results. The methods are suitable for studies of clonal relations in an evolutionary sense rather than clonal spread in an outbreak situation. Advantages are lack of biased results, easy interpretation and possibilities of exchange of data via the internet MLST. Recently, an MLST scheme was developed for E. faecium and typing results suggest that epidemic lineages of E. faecium emerged worldwide and that certain such lineages have the ability to persist and colonize patients in hospitals. These lineages (only VRE) have almost uniformly harbored a variant esp gene, suggested to be associated with colonization and possibly with increased virulence in these bacteria.

Phenotypic methods

These methods are based on the phenotypic expression of genes rather than the sequences of these and are cheaper and often simpler to perform but not as sensitive as the genetic methods. The growing interest in ecological investigations, when often large number of isolates need to be typed, has resulted in a need for faster and cheaper typing techniques for bacteria. The PhenePlateTM RF (PhP-RF) system is a recently developed phenotypic method, based on a 96 well microplate containing 8 sets of eleven dehydrated reagents, selected to have a high discriminatory power among enterococcal isolates. The kinetics of each reaction is evaluated by measuring the absorbance value of each well three times during 64 hours, and a biochemical fingerprint is calculated as the mean value for each reagent over the three readings. The PhP-RF method was shown to be highly reproducible, even when results from different laboratories were compared, and the discriminatory power, measured as Simpson’s diversity index, was as high as 0.96 for all enterococci.