Species identification of enterococci by biochemical test and molecular-genetic methods

Monika Lavová,Konrad Domig,Jana Bezeková,Miroslav Kročko,Margita Čanigová

doi:10.5219/364

Abstract

The aim of this study was comparison different methods of species identification of enterococci. One hundred and fifty three suspected colonies were isolated from milk and dairy products (cheeses from cow´s, ewe´s and goat´s milk). On the bases of their growth on BEA agar, microscopic characteristic, results of Gram staining, catalase test and PYRAtest was thirty four isolates assigned to the genus Enterococcus. These isolates were identified by commercial biochemical test EN-COCCUS. 52.9% of them were included in species E. faecalis, 29.4% in E. faecium, 14.7% in E. durans and 2.9% in E. group III. This group includes 3 species: E. durans, E. hirae, E. faecalis asaccharolytic var. Then 16S rRNA sequencing nucleotide of all isolates was realized. Results of sequencing were compared with NCBI database. Only 14.7% of isolates were in 100% accordance. One from them was species E. durans and others were designated as E. faecium. For 20.6% of detected isolates was in accordance with more reference strains. Other isolates were identical with reference strain on 99%. For verification of all results species-specific PCR was used and 52.9% isolates were identified as species E. faecalis, 32.4% as E. faecium and 14.7% as E. durans. Strains belonging to the species E. faecalis were identified the most reliable by all used methods.

Highlights

Enterococci are frequently associated with many foods from animal and vegetable origins (Krebs-Artimová et al, 2013)
DNA of enterococci was isolated by peqGOLD Bacterial DNA Kit (Peqlab, Germany) and concentration and purity was detected on spectrophotometer NanoDrop 2 000c spectrophotometer (Thermo Scientific, Germany)
Same mixture was used for identification of E. faecalis and E. faecium

Summary

Introduction

Enterococci are frequently associated with many foods from animal (dairy and meat products) and vegetable origins (Krebs-Artimová et al, 2013). The objective of the present study was to evaluate accuracy of identification molecular and biochemical methods for enterococci isolated from raw cow milk and dairy products. Results of EN-COCCUS test were confirmed by 16S rRNA sequencing and species-specific PCR methods. The PCR method for the species identification of enterococci isolates were performed using specific primers (Table 1).

Objectives

Results

Conclusion