How does enterococcus faecalis obtain energy

However, E. Proteomic analyses with systematic exposure to various stresses have previously identified six genes encoding general stress response proteins GSPs which were up-regulated in E.

The reasons for the contrary results may be that the time of E. In this study, de novo transcriptome sequencing of E. A total of 18,, high-quality transcriptome reads were obtained, giving rise to an average of bp per read. Compared to the control group, many genes involved in carbohydrate transport and metabolism and energy production and conversion were significantly down-regulated, but genes involved nucleotide and amino acid transport and metabolism were significantly up-regulated, which might contribute to adaptation and survival of E.

Most interesting is mostly differently expressed stress response genes were down-regulated in alkaline stress. This study provides new insights into the adaptive process of E. It is hoped that future studies can incorporate the rapid advances underway in metabolomics and proteomics. Extrapolation of the present data to the clinical situation can only be speculative. However, the present data may contribute toward better understanding the survival strategies of E.

The draft genome sequence of E. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Table S1. KEGG summary of E. Table S2. The detail sequence information of the transcriptome. Table S3. The data of all of the gene transcription information XLS. Table S4. The list of genes found to be induced up-regulated in alkaline stress.

Table S5. The list of genes found to be decreased down-regulated in alkaline stress. Aakra, A. Transcriptional response of Enterococcus faecalis V to erythromycin. Agents Chemother. Appelbe, O. Effects of prolonged exposure to alkaline pH on Enterococcus faecalis survival and specific gene transcripts. Oral Microbiol. Aslangul, E. Acquired gentamicin resistance by permeability impairment in Enterococcus faecalis.

Athanassiadis, B. The use of calcium hydroxide, antibiotics and biocides as antimicrobial medicaments in endodontics. Bizzini, A. Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis. Bourgogne, A. Genome Biol. Burgos, M. Multilocus sequence typing of Enterococcus faecalis from vegetable foods reveals two new sequence types.

Foodborne Pathog. Bystrom, A. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Camacho, C. BMC Bioinform. Conesa, A. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21, — Creti, R. Survey for virulence determinants among Enterococcus faecalis isolated from different sources.

Deibel, R. Utilization of arginine as an energy source for the growth of Streptococcus faecalis. PubMed Abstract Google Scholar. Deutscher, J.

How phosphotransferase system-related protein phosphorylation regulates carbohydrate metabolism in bacteria. Distel, J. Biofilm formation in medicated root canals. Evans, M. Mechanisms involved in the resistance of Enterococcus faecalis to calcium hydroxide. Fisher, K. The ecology, epidemiology and virulence of Enterococcus.

Microbiology , — Flahaut, S. Alkaline stress response in Enterococcus faecalis : adaptation, cross-protection, and changes in protein synthesis. Freeman, B. Biology of disease: free radicals and tissue injury. Ghim, S. Giard, J.

Glucose starvation response in Enterococcus faecalis JH survival and protein analysis. Inactivation of the stress- and starvation-inducible gls24 operon has a pleiotrophic effect on cell morphology, stress sensitivity, and gene expression in Enterococcus faecalis. Gomes, B. Enterococcus faecalis in dental root canals detected by culture and by polymerase chain reaction analysis.

Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Gupta, S. Purification and characterization of guanylate kinase, a nucleoside monophosphate kinase of Brugia malayi. Parasitology , — Haapasalo, H. Inactivation of local root canal medicaments by dentine: an in vitro study. Halliwell, B. Oxidants and human disease: some new concepts. Hames, C. Glycerol metabolism is important for cytotoxicity of Mycoplasma pneumoniae. Heefner, D. Imlay, J. DNA damage and oxygen radical toxicity. Science , — Kakinuma, Y.

Inorganic cation transport and energy transduction in Enterococcus hirae and other streptococci. FEBS Lett. Kamat, S. The catalase activity of diiron adenine deaminase. Protein Sci. Kayaoglu, G. Growth at high pH increases Enterococcus faecalis adhesion to collagen. Koo, B. Enhancement of thymidine production in E. Langmead, B. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Despite this, infection mechanisms, especially the transcriptional modulation occurring in living hosts, are still poorly understood.

In this study, we analyzed the role of citrate fermentation in the pathogenicity of E. Citrate metabolism deficient- E. We also found that an active citrate metabolism allows E. Duplicated genomes were detected and removed prior to analysis. Proteins belonging to E. Cut-off values for searches carried out in E. Bacterial strains and plasmids used in this study are listed in Table 1.

For urine or blood growth experiments, E. When an OD value of 0. The E. After digestion, the PCR product was ligated into the corresponding sites of the pGhost9 vector. The resulting plasmid, named pmCit Table 1 , was introduced into E.

Briefly, E. Correct amplification of oadA was confirmed by sequencing. The plasmid bearing the promoter-gfp and -cherry transcriptional fusion is derived from the pTLGR plasmid [ 29 ]. Fragment orientation was determined by PCR, and confirmed by sequencing. Then, the bacterial cultures were diluted in 50 ml of LB medium to a final OD of 0.

Larvae were inoculated by direct injection into the hemocoel using a Hamilton syringe equipped with a repeating dispenser. Survival of the individuals was monitored every two to four hours, by direct observation and gently touching non-motile larvae to evidence movement response or confirm death. Survival of the larvae group was evaluated until 72 h post-infection. Fifteen larvae were separated from the group at 0, 24 and 48 h post-inoculation, three sub-groups for each time were formed hemolymph extracted, pooled together and suspended in cold Insect Physiological Saline IPS buffer mM sodium chloride, 5 mM potassium chloride, 10 mM Tris HCl pH 6.

The assay was carried out in duplicate, with three technical replicates for each time point. At different times, hemolymph was extracted and pooled from larvae inoculated with E. Fiji software [ 34 ] was used to pseudocolor images. Data analysis was done using R software. Survival curves were constructed according to the Kaplan-Meier method, using the LogRank and Holm-Sidak tests [ 35 ] for multiple comparisons. In our model the fixed effect are the strains and random effect is the time.

For the hemolymph experiment the experimental unit is the pool of 5 larvae; for the experiment with blood and urine the experimental unit is each tube where bacteria was grown. P value was set at 0. A total of genomes comprising different enterococci species were selected for further analysis. Using the citrate lyase citE and citF genes from E. Consequently, we focused the search on the E. From available genomes, we found that citrate metabolism is present in almost all of them, since genomes encoded the citrate lyase genes.

Next, all the genes of the cit cluster in the E. Gene context analysis of Enterococcus genus representative strains revealed that Type I citrate metabolism cluster is present only in E. On the other hand, Type II was found in at least fifteen species: E. These results suggest that this metabolism could be playing an important role in these microorganisms ability to grow, persist or colonize different niches. In particular, the genes and arrangement described for the E.

In all of the E. The connection between citrate metabolism and aroma compound production pathways has been extensively studied as well as the regulatory mechanisms involved [ 8 , 18 — 22 , 37 ].

Although many E. Thus, G. Thus, to determine if genes responsible for citrate fermentation are expressed in E. Fluorescence microscopy at different time points of E. Representative individuals of inoculated larvae are shown in C. Two independent experiments were carried out and representative images acquired are shown. Next, E. No fluorescence was detected in the empty pTLGR plasmid not shown.

Next, G. Hemolymph of sample individuals was extracted at different time points after inoculation and fluorescent bacteria were detected. After 24 hours of inoculation Fig 2C , evidence of active cit promoters was found, both free in the hemolymph and associated to hemocytes.

A strong fluorescent signal is observed around the hemocytes, indicating that several bacteria are in contact with them, at 48 h. Also during the assay, melanization and deterioration of larvae health conditions was observed for individuals inoculated with E. Production of melanin by G.

In our case, distinctive dark spots were observed after 24 h of infection while at 48h melanization extended to the rest of the larva as a consequence of infection progression. No fluorescence was detected in the hemolymph of larvae inoculated with E. Taking into consideration the above data, contribution of the cit cluster to E.

Larvae group survival was monitored up to 72 h post-infection and resulting data were analyzed through Kaplan-Meier curves [ 35 , 42 ]. No mortality was observed in PBS-injected G. Larvae inoculated with E. A Kaplan-Meier survival plots of G. F Images of innoculated larvae showing different degrees of disease.

Next, citrate fermenting-deficient E. Despite the low survival percentage observed for the wild type WT strain, almost no larvae mortality was obtained over the course of a 72 h experiment for the JHCit - strain Fig 3A. Remarkably, these low lethality levels are similar to those observed with the nonpathogenic innocuous L.

These findings suggest that larvae mortality induced by E. In order to extend our knowledge about the role of citrate metabolism in E. The JHOadA - strain cannot metabolize citrate beyond oxaloacetate; on the contrary the JHOadB - strain is capable of slowly degrading oxaloacetate to pyruvate by the action of the cytoplasmic OadAHD complex [ 21 ]. Thus, survival of G. Since lethality is influenced by inoculum concentration, we injected larvae with several CFU values to observe variations in strain pathogenicity.

Fig 3C shows the results for the 1 x 10 7 CFU inoculum. To corroborate the role of citrate metabolism in E. Repizo et al [ 21 ] showed that the JHOadA - strain cannot metabolize citrate and, consequently, does not show growth improvement in the presence of this compound.

On the other hand, in LB growth medium supplemented with citrate, JHOadB - is able to reach final OD levels similar to those of the parental strain, with a delay in the beginning of the second growth phase, i. Thus, despite the ability of JHOadB - to grow in citrate-containing media LBC , it seems that under infection conditions, the delay observed in its growth is strongly detrimental for the cells and they cannot cope with the immune system of G.

This leads to higher larvae survival rates. Nonetheless, citrate metabolism deficiency makes strains less virulent than the WT, confirming the observation made for the JHCit - strain. As a consequence, this strain exhibits growth deficiency in the presence of pyruvate at pH 5. This indicated that the pathway involved in aroma compound generation is an important mechanism which allows growth in acidic media [ 37 ].

After examination of larvae health status during 72h, KM curves were plotted Fig 3E. In previous sections we showed that citrate metabolism plays an important role during E. Consequently, an analysis of cit cluster induction in other animal fluids associated to common diseases caused by E. To this end, E. GFP and Cherry fluorescent signals were detected as early as 2 hours after exposition to blood Fig 4A , indicating that both promoters were quickly induced. When E. These results suggest that citrate metabolism in E.

To confirm this hypothesis in G. Larvae groups were infected with the WT and citrate metabolism-mutant strains. Two independent experiments were carried out and representative images acquired of three technical replicates are shown. Bacterial burden was quantified using pools of hemolymph extracted from different larvae at the time points indicated.

B and C Growth of E. These results suggest that citrate metabolism-deficient strains are unable to proliferate as well as the WT in the hemolymph and are probably eliminated by the larvae immune system, thus provoking a less aggressive infection.

Since we showed that citrate genes are induced in blood, we analyzed the growth behavior of the various mutants and the OadA - complemented strain to determine if their differential capacity for citrate utilization can be correlated with a differential growth. But, after 24 h of growth, E. The strains of E. It interacts with many other organisms and has effects on the environment. The Enterococci are members of the bacterial community inhabiting the large bowel in humans.

They also are a natural part of the intestinal flora in most other mammals and birds 8. The Enterococci are also found in soil, plants, and water.

When they are found in water it is typically because the water had been contaminated with fecal matter. Although E. The ecology of antibiotic resistance and virulence gene transfer in the environment is still not well understood.

Insects, such as houseflies HF , that develop in decaying organic material can transmit antibiotic-resistant bacteria from the manure of animals and other decaying organic substrates to residential settings 7. HF are perfect transmitters because of the live microbial communities present in the habitats where they develop e. Adding to the good transfer qualities are the way in which HF feed their young regurgitation and their attraction to human food.

Since HF can fly long distances, this insect is very good for spreading fecal bacteria, including human and animal pathogens, and possibly antibiotic-resistant strains of Enterococci 7.

A recent study screened for antibiotic resistance and virulence genes in Enterococci from HF in fast-food restaurants in Kanasas. The effects that E. They typically contaminate water supplies that can lead to infected plants as well as infections in people 8.

The antibiotic factors can also be transported by various insects e. Enterococci have emerged as a major cause of nosocomial infections, and within this group Enterococcus faecalis causes the majority of human enterococcal infections. These infections may be local or systematic and include urinary tract and abdominal infections, wound infections, bacteremia, and endocarditis 2.

This can mean serious health problems, which include the lack of available antibiotic therapy for VRE infections, because most VRE strains harbor resistance to multiple antibiotics besides vancomycin e.

The transfer of vanocmycin resistant genes from VRE to other Gram-positive pathogens is a serious public health concern. The most common way that the E. Enterococci can be carried on the hands of health care workers and be carried transferred from one patient to another.

It has been shown that VRE on the hands can persist for up to 60 minutes 8. Rectal thermometers, not properly cleaned after use, can transmit the VRE from patient to patient as well. The acquired strain, carrying antibiotic resistance genes, is able to live in the GI tract.

Infections then arise from these newly acquired E. The most common infection caused by Enterococci is infection of the urinary tract. The source of bacteremia is most often the urinary tract, occurring from an infected intravenous catheter. Endocarditis is the most serious enterococcal infection, as it causes inflammation of the heart valves. In many cases of endocarditis, antibiotic treatment fails and surgery to remove the infected valve is necessary 8.

Less common infections caused by E. Due to the resistance of Enterococci to many antibiotics, treatment of these infections is difficult. Enterococci have been studied for possible use as a probiotic a dietary supplement that contains living non-virulent microbial cells that when ingested are thought to beneficially affect the composition of the intestinal microflora.

Administration of the E. Due to the high disease causing properties of E. It is known that vancomycin-resistant bacteria e. The enterococcal infections are challenging because the organisms have the ability to quickly acquire and disseminate resistance genes. Ceftobiprole BPR was used as an investigational cephalosporin against Gram-positive cocci. BPR is a broad-spectrum parenteral cephalosporin with high affinities from Gram-positive and Gram-negative penicillin-binding proteins.

This study examined the activity of BRP against a large collection of E. The study found that susceptibly to BPR in E. In the strains of E. The researchers showed that BPR exhibited bactericidal activity against E. This research demonstrated that BPR has potent activity against a very large collection of E. When antibiotics are used in the treatment of a bacterial infection, they can have an impact on the intestinal flora.

Resistant bacteria can be selected during treatment, such as the Enterococci , and are potentially pathogenic. The emergence of resistance is an issue for new antibiotics because it could risk the usefulness of antibiotics.

This study examined the emergence of resistance to antibiotics by E. Linezolid can be used against multiple-drug-resistant Gram-positive cocci, including VRE. It inhibits bacterial protein synthesis by binding specifically to a domain in the 50S ribosomal subunit and is not affected by the resistance mechanisms that affect other antibiotics.

This study looked at the rate of emergence of linezolid-resistant E. The does of linezolid was fed in water with a doses varying from 0.

The mutants were all dependent on the linezolid given, levels of resistance increased with the duration of exposure. No mutants were isolated in the absence of linezolid, suggesting that de novo resistance to linezolid was uncommon in the Enterococci.

The research found that a mutation in a single 23S rRNA gene was the critical step in the emergence of linezolid resistance. Primary colonization with single-mutation mutants were observed as early as 5 days after treatment initiation in mice.

These experiments involving mice help explain the pattern of emergence of resistance to linezolid observed in clinical isolates.