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Antibiotic Stress and Virulence of Enterococci


Stress by Antibiotics and Virulence of Enterococci







The SAVE Team



Opportunistic pathogens constitute a very interesting and challenging area of research. That is the case of bacteria from the genus Enterococcus. These gut commensals are also found in food products, water, sand and soil, therefore maintaining with humans and animals a very tight and happy coexistence. However, in the last decades, due to the increased use of antibiotics, both in animal breeding and in Hospitals and veterinary clinical practice, enterococci became a major concern in terms of nosocomial and community acquired infections. This urged us to find new ways of dealing and controlling enterococcal infections, thus directing our research into three main topics:




Stress response


Antibiotic Resistance








Enterococcus are intrinsically resistant to many of the antibiotics used in clinical practice. Moreover, they have acquired resistance to other antibiotics, with the consequent appearance of multiresistant strains, turning treatment of these infections very difficult in many cases.



Particularly worisome is the acquisition of vancomycin resistance in enterococcal strains. Vancomycin is considered a last resource antibiotic in the treatment of multiresistant enterococcal strains and is also used to treat infections caused by other pathogens like Staphylococcus, Streptococcus and Clostridium. After the introduction of this antibiotic into the clinical practice in the 80s, the first report of VRE (Vancomycin-Resistant Enterococcus) appreared soon in 1986. Vancomycin resistance is now spread both in the Hospital settings and in other environments. Vancomycin resistance in Enterococcus is generally acquired and is encoded in genetic mobile elements, like transposons. It can be associated to several genotypes, although the most frequently found are VanA and VanB, conferring inducible resistance to vancomycin. Vancomycin is a cell-wall targeted antibiotic. Using both proteomic and transcriptomic approaches we realized that both VRE and  VSE (vancomycin-susceptible enterococci) respond to this cell-wall acting antibiotic through specific two-component systems and other major regulators in the cell.



Antibiotic use as growth promoters in animal feeding is considered one major factor responsible for the increase of infections caused by multiresistant enterococci. Between 1997 and 1999 some growth-promoting antibiotics, namely bacitracin, were withdrawn as such by the EU on the basis of the Precautionary Principle. Bacitracin is scarcely used in the Hospital setting and in agriculture and was banned from animal feeding in Europe almost ten years ago. However, recent work reported the spread of high levels of bacitracin resistance, which could point to cross-resistance to other in use antibiotics. We have found a new genotype of bacitracin resistance, which is spread in several species and environments and we are currently studying its regulation and the correlation between genotype and phenotype (MIC values).


Resistance in the environment

Due to the flow of both enterococcal strains, and consequent trading of genes encoding resistance to antibiotics, between several environments, it is important that the spread of both antibiotic resistance and resistant strains is kept under surveillance. Therefore, we collect enterococcal isolates from different environments (food, hospital, community, pets, water, and sand) type them, using molecular techniques (PFGE and MLST), and characterize them concerning antibiotic resistance (MIC determination and detection of resistance genes) and virulence potential.


Understanding the intrinsic resistance of Enterococcus

Intrinsic resistance to antibiotics is either due to the absence of target (or decreased affinity to the target), or to efficient extrusion of the antibiotic molecule out of the cell or even due to impossibility of the antibiotic to reach the target. We are involved in an Era-Net Pathogenomics Project in which, among other objectives, we are trying to target the genes responsible for the intrinsic resistance of E. faecalis to many antibiotics.



A biocide is an active substance within a formulation used for antisepsis or surface disinfection. There is a large number of both biocides and of formulations. In contrast to antibiotic resistance, which is well documented, bacterial resistance to biocides has only been reported recently, and only a few biocides have been studied. Moreover, recent evidence suggests that some mechanisms can confer resistance to both biocides and antibiotics. We are currently studying biocide susceptibility of strains from several origins trying, in parallel, to find the mechanisms underlying biocide resistance in Enterococcus.




The dual lifestyle of Enterococcus as both commensal and pathogenic organisms requires them to be responsive to varying environmental conditions, both in and outside the host. Its ability to sense changing environmental stimuli and respond accordingly is of the utmost importance in its adaptation to these varying conditions. A major question to be answered is how Enterococcus accomplishe such a feat.

The responses of E. faecalis to some environmental stresses have been studied during the last decade and have shown its exceptional ability to survive and persist in a variety of adverse environments. Indeed, E. faecalis cells can resist stresses such as heat, high osmolarity and presence of ethanol, detergents, hydrogen peroxide, sodium hypochloride and heavy metals. However, there are not clues on which message is perceived by the cell upon imposition of the stress, either acid, oxidative, osmotic, heat or other, and on how the outside message is perceived on the inside. We are interested in identifying some of those signals that show a marked up or downshift in transcription of the genes already described to be involved in stress responses and their respective role in the overall cellular processes. This will be accomplish using DNA microarray, measurement of membrane associated parameters, such as DpH and pmf, ATPase activity and aminoacid transport and 2D- gel electrophoresis of proteins. In particular, we are interested in metal ion stress (some of these ions have been implicated also in virulence in other bacteria) and in sub.inhibitory concentrations of antibiotics.





The ability to cause infections in Hospitals has been associated with several virulence factors carried by E. faecalis. They are involved in attachment both to host cells and to extracellular matrix proteins, in resistance to macrophages, in cell and tissue damage and in immune system evasion. Following adhesion to host cell surfaces, and evasion of the host immune response, the last step in the pathogenesis of infection is the production of pathologic changes in the host. Enterococci virulence is still debated and puzzling, not only because both infectious and non-infectious strains carry the same virulence traits, but also because the mediators of virulence may be diverse between strains.

Host changes can be induced by direct tissue damage as a result of secreted proteases, namely gelatinase. This metalloprotease is encoded in gelE gene, which is under the regulation of the fsr operon. Fsr is a quorum-sensing system, similar to S. aureus Agr system. However, it is much less studied and understood. Both fsr and gelE have been clearly implicated in E. faecalis virulence using several animal models. These virulence factors are disseminated in the genus Enterococcus and have been reported in non-clinical strains, namely from food, and in other species, namely E. faecium and E. durans. We are studying the implications of the presence of these virulence factors in food strains and in the non-E. faecalis strains, where they appear to have a different impact in virulence. Many investigators have reported the absence of gelatinase activity in the presence of the complete genotype fsr-gelE, which is another subject under investigation at the moment, together with the regulation of the fsr operon, which is poorly understood.

In the Era-Net Pathogenomics Project, in which we are partners, a mutant library of E. faecalis is being produced, targeting genes that might be involved in the transition from commensalism to pathogenicity in this bacterium. These mutants will, among other studies, be tested for their ability to survive in macrophages.

One of the key players in bacterial virulence is certainly RNA, in particular the ncRNA, which have been reported in other genus to modulate virulence. Their role in Enterococcus virulence is also under study in our Lab.





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