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Journal of Clinical Microbiology, June 2003, p. 2569-2576, Vol. 41, No. 6
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.6.2569-2576.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Comparative Study Using Type Strains and Clinical and Food Isolates To Examine Hemolytic Activity and Occurrence of the cyl Operon in Enterococci

Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisbon,¹ Instituto de Biologia Experimental e Tecnológica and Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, 2781-901 Oeiras,² Estação Agronómica Nacional, Instituto Nacional de Investigação Agrária, Quinta do Marquês, 2784-505 Oeiras, Portugal³

Received 15 October 2002/ Returned for modification 17 December 2002/ Accepted 26 February 2003

The hemolytic ability, the presence of cyl genes, and the diagnostic accuracy of cytolysin molecular detection were investigated in the genus Enterococcus by using 164 strains from 20 different species (26 reference strains, 42 clinical isolates from human and veterinary origin, and 96 isolates from ewe cheese and milk). Hemolysis was assayed with sheep and horse erythrocytes and under aerobic or anaerobic conditions. Screening of cytolysin genes (cylL_L, cylL_S, cylM, cylB, and cylA) was performed with new specific primers and the anaerobic assay of beta-hemolysis was used as the "gold standard" for the evaluation of cyl gene-based PCRs. Since beta-hemolysis and cyl genes were found in 10 and 14 species, respectively, the hemolytic ability seems to be spread throughout the genus Enterococcus. Beta-hemolysis was observed in 6 of 26 (23%) reference strains, 14 of 42 (33%) clinical isolates, and 6 of 96 (6%) food isolates. The presence of cyl genes was detected in 15 of 26 (58%) reference strains, 37 of 42 (88%) clinical isolates, and 67 of 96 (70%) food isolates. These data indicate a virulence potential in food isolates, reinforcing the need of their safety assessment. Analysis of phenotypic-genotypic congruence suggests a divergent sequence evolution of cyl genes and the effect of environmental factors in the regulation of cytolysin expression. Evaluation of the diagnostic accuracy of cytolysin molecular detection points to cylL_L-based PCR and cylL_LL_SMBA-based PCR as the most reliable approaches. Nevertheless, the low sensitivity (46%) and gene variability indicated by our study strongly recommend the phenotypic assay for the assessment of hemolytic ability in enterococci, followed by the molecular screening of cyl genes in nonhemolytic strains to evaluate their virulence potential.

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