Virulence patterns of Enterococcus spp. associated with human pathology of marine and clinical origin isolated in Lima, Peru. Rojas Favio1, Alvarado Debora1, ...
Virulence patterns of Enterococcus spp. associated with human pathology of marine and clinical origin isolated in Lima, Peru. Rojas Favio1, Alvarado Debora1, Rosas Kristel1, Huamán Ana1, Ramírez Rafael2. 1Laboratorio
de Microbiología Molecular y Biotecnología, Facultad CC.BB.-UNMSM 2Departamento de Patología Clínica - Hospital Guillermo Almenara Irigoyen FigurE. Multiplex pcr for virulence genes in marine and nosocomial enterococci
Introduction: The sea of Lima and Callao, highly impacted by domestic, industrial and hospital receives an average discharge of 10.91 m3 /s of untreated wastewater (salbatier et al., 2008). While Enterococcus, considered a few years ago as a non-pathogenic commensal organism, has a rapid molecular evolution and has acquired a wide variety of virulence markers. Currently, enterococci are responsible for multiple infections, including intra-nosocomial infections, but it is unknown virulence profile that present locally. Given its ability to store and transfer genetic information, the persistence and increase of these markers in the marine environment would imply a potential risk to public health.
In nosocomial isolates, a high frequency of phenotypic virulence determinants of E. faecalis and E. faecium was observed, while in E. hirae, durans, rafinosus avium did not showed these determinants; all strains of E. faecalis were strong producer of biofilm, 81.8% shows gelatinase activity, while 27.3% hemolytic phenotype. In the case of E. faecium only 5.5% were positive for the 3 phenotypic determinants. Biofilm production appears no to be associated with esp gene. There is a greater diversity of virulence markers in nosocomial isolates against the marine isolates, while hyl and cylA genes were not detected among marine isolates. the profiles gelE+esp+, esp+ and gelE + are reported in this investigation. All selected markers were frequently founded in clinical Figure 1. Lane 1,7: mix PCR (cylA; esp; hyl; GelE); lane 2: E. faecium marine esp+gelE+; nosocomial strains: lane 3: E. faecalis esp+gelE+; lane 4: E. isolates, however the hyl+ marker was present only in vancomycin resistant faecalis gelE+; lane 5: E. faecalis cylA+gelE+ E. faecium (VRE). Table 1 and 2 shows the prevalence of virulence markers in nosocomial and mairine enterococci.
Objectives: Establish profiles of phenotypic and molecular determinants of virulence in Enterococcus spp. associated with human pathology of marine and nosocomial origin in Lima, Peru. Methods: Thirty two strains of marine enterococci were selected (Lima´s bay): E. faecalis (5), E. faecium (9), E. durans (9) and E. hirae (9), and 33 nosocomial strains were isolated at the Hospital Nacional Guillermo Almenara: E. faecalis (11) and E. faecium (18), E. durans, E. hirae, E. rafinosus and E. avium (1 each one).
Hemolysin test was performed on Todd-Hewitt agar suppl. with 5% defibrinated erythrocytes (Arularasi et al., 2011). Gelatinase was determined according to Pires-Boucas et al., (2010). Biofilm assay was performed in polystyrene microtiter plates and stained with 1% safranin; the absorbance of biofilm was determined at 492 nm (Jayanthi et al. 2008). The detection of virulence genes hyl, cylA, esp and gelE were performed by multiplex PCR (Vankerckhoven et al., 2004).
Results: In marine strains, beta-hemolytic activity was not observed, one strain of E. faecalis produced gelatinase only, while there is a strong biofilm production in 60% of E. faecalis, in 11.1% of E. faecium, in 22% of E. durans and in 33.3 % of E. hirae.
Conclusions: Our observations show a low presence of virulence markers in marine enterococci and a greater number of different profiles of virulence in nosocomial E. faecalis and E faecium in contrast with marine strains. We found that in marine environment, certain genetic markers no necessarily are associated with expression of virulence determinants. Table 1. Prevalence of of single/multiple virulence markers in nosocomial enterococci Combination of virulence-marker/s
Total enterococci n(%)
E. faecalis
cylA+/esp+/gelE+
3 (9.09)
3 (9.09)
gelE+/ esp+
3 (9.09)
Hyl+/ esp+
E. faecium
Table 2. Prevalence of of single/multiple virulence markers in marine enterococci
Others enterococci
Combination of virulence-marker/s
Total enterococci n (%)
E. faecalis
-
-
gelE+/ esp+
1 (3.16)
-
1 (3.16)
-
-
2 (6.06)
1 (3.03)
-
gelE+
5 (15.63)
2 (6.25)
1 (3.13)
2 (6.25)
-
2 (6.06)
-
2 (6.06)
-
esp+
2 (6.25)
2 (6.25)
-
-
-
gelE+
8 (24.24)
5 (15.15)
3 (9.09)
-
cylA+
-
-
-
-
-
esp+
1 (3.03)
-
1 (3.03)
-
Hyl+
-
-
-
-
-
cylA+ Hyl+
1 (3.03)
-
1 (3.03)
-
E. faecium
E. durans
E. hirae
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