DISCRIMINATORY IDENTIFICATION OF Enterococcus faecium AND Enterococcus faecalis ISOLATED FROM A RICOTTA PROCESSING BY DNA SEQUENCING

Enterococcus genus is characterized by the high variability of ITS region, and the isolates have regions with different sizes, allowing the differentiation of Enterococcus at the species level. The ITS 16S-23S region, therefore, is a good candidate for the differentiation between bacterial species of this genus. The objective of this work was to differentiate strains of E. faecium and E. faecalis isolated from the ricotta processing line by sequencing the ITS 16S-23S region. To identify potential contamination sources during ricotta processing, 57 E. faecium cultures and 50 E. faecalis cultures were subjected to discriminatory identification to differentiate genetic profiles of each strain. The amplification of ITS region of 16S-23S rDNA was performed using PCR. The amplicons generated by ITS-PCR were subjected to sequencing in an automatic capillary sequencer. The sequences obtained were compared with the sequences available in Genbank. The multiple ITS sequences were aligned using Clustal W software integrated with MEGA5 software. The phylogenetic tree was constructed using the neighbor-joining, maximum-parsimony, and maximum likelihood algorithms present in these software programs. According to ITS-PCR analyse, four different clusters could be identified, and 27.1% (29/107) of the isolates belonged to cluster I, 7.5% (8/107) to cluster II, 64.5% (69/107) to cluster III and 0.9% (1/107) to cluster IV. Thereafter, sequencing of ITS region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, most of the E. faecalis isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other. The same was observed for E. faecium isolates. For E. faecalis, only strains 012 and 015 (from packaged ricotta in collection 1) and strains 061 and 062 (from packaged ricotta in collection 3) had genetic similarities regarding the ITS and the same virulence profile. For E. faecium, only strains 053 and 057 (from packaged ricotta, collection 3), and strains 002 and 103 (from packaged ricotta and molding countertop, respectively) had genetic similarities regarding the ITS and the same virulence profile; thus, they may be considered as the same strain. The similarity between strains 002 and 103 indicates the molding countertop as a possible source of E. faecium contamination of the ricotta. The results also revealed a great deal of genetic diversity among E. faecalis and E. faecium isolated from the ricotta processing line.