Comparative study using type strains and clinical and food isolates to examine hemolytic activity and occurrence of the cyl operon in enterococci.
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(L)L(S)MBA-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.
Semedo
Teresa
T
Departamento de Biologia Vegetal and Centro de Genética e Biologia Molecular, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisbon, Portugal.
Almeida Santos
Margarida
M
Martins
Paula
P
Silva Lopes
Maria Fátima
MF
Figueiredo Marques
José J
JJ
Tenreiro
Rogério
R
Barreto Crespo
Maria Teresa
MT
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002611:Cheese / Q000382:microbiology; D003603:Cytotoxins / Q000235:genetics* / Q000378:metabolism; D016983:Enterococcus / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000472:pathogenicity; D016908:Gram-Positive Bacterial Infections / Q000382:microbiology; D006460:Hemolysin Proteins / Q000378:metabolism*; D006801:Humans; D008892:Milk / Q000382:microbiology; D009876:Operon; D010641:Phenotype; D016133:Polymerase Chain Reaction
D001426:Bacterial Proteins; D003603:Cytotoxins; D006460:Hemolysin Proteins