Contribution of citrate metabolism to Enterococcus faecalis virulence

MAGNI, C; MARTINO, G; BLANCATO, VS

San Miguel de Tucumán

Simposio; V Simposio Internacional de Bacterias Lácticas (SIBAL); 2016

CERELA

Enterococci are present as non-starter lactic acid bacteria in a wide variety of cheeses, mostly produced from raw milk. These microorganisms contribute during the ripening process of cheese to the development of organoleptic properties; but they can be potentially hazardous to human health due to their pathogenicity. Enterococcus faecalis citrate metabolism seems to be an example of this duality. Citrate degradation end products (diacetyl, acetoin and butanediol) improve the aroma of chesses; on the other hand, microarray data suggested that E. faecalis citrate metabolic genes (cit genes) could be up-regulated in blood and urine. Furthermore, our previous virulence analysis of E. faecalis cit deficient strains in Galleria mellonella insect model revealed a lower lethality when compared with the E. faecalis wildtype strain (JH2-2).In this work we analyzed E. faecalis growth in correlation with the activation of cit genes in blood, urine and G. mellonella. When incubated in blood JH2-2 and citrate liase mutant (CL-) strains showed similar behavior reaching the same final CFU/ml, nevertheless fluorescence microscopy allowed to detect induction of the cit promoters. Citrate concentration in blood is very low (less than 190 µM) probably not enough to increase the growth of the wildtype strain. On the contrary, in urine a clear difference was observed; JH2-2 strain reached higher CFU/ml than CL- strain, accordingly induction of the cit promoters was detected. In urine citrate concentration is around 1-2 mM allowing a stronger induction of the promoters and consequently better growth of the wildtype strain able to metabolize it.In G. mellonella hemolymph citrate is also present at low concentrations. To prove if there is a correlation between the differences observed in G. mellonela killing and E. faecalis growth in hemolymph, we inoculated groups of larvae with CL- and WT strains at the same initial CFU/individual. Hemolymph was extracted at different times and total CFU/ml of hemolymph was determined, resulting in a marked difference between the two strains. CL- inoculated larvae have lower CFU/ml 48h post inoculation, meaning that this strain is unable to grow as well as the WT, probably causing the observed less virulent infection.In order to observe cit promoter induction in G. mellonella, its hemolymph was extracted at different time points after injection with JH2-2 harboring reporter plasmid. In this insect model, fluorescent bacteria were detected after 24 hours of inoculation, both free in the hemolymph and associated to haemocytes (insect immune cells).Our previous and present results suggest that citrate metabolism, a desired technological trait in Enterococci could also, in certain conditions contribute to its virulence.