The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.     

Conjugal transfer of bacteriocin plasmids from different genera of lactic acid bacteria into Enterococcus faecalis JH2-2

The lactic acid bacteria (LAB) are of great interest because of their food grade quality and industrial importance. In the recent past, the pediocin PA-1 like bacteriocin was found to be synthesized in cross-species and cross-genera. Hence, the present work was carried out in order to determine the transfer of plasmid encoded pediocin PA-1 like bacteriocin among LAB. The objective of this study is to demonstrate the dissemination of bacteriocin-encoded plasmid from Pediococcus acidilactici NCIM 5424, Enterococcus faecium NCIM 5423 and Lactobacillus plantarum Acr2 to Enterococcus faecalis JH2-2 under in vitro (filter mating method) and in situ (soymilk model) conditions. The fermentation of the soymilk was determined by the selected pediocin producers. E. faecium NCIM 5423 was able to transfer the bacteriocin only under in situ conditions, whereas the native pediocin producer P. acidilactici NCIM 5424 transferred the bacteriocin under both the methods used. The in situ method gave more transfer frequency, ranging from 10^?7 to 10^?4 transconjugants per recipient cell. No conjugal transfer by L. plantarum Acr2 was observed. The physiological conditions like pH and temperature were found to influence the production of bacteriocin in the obtained transconjugants. The results suggest the horizontal gene transfer (HGT) and the natural spread of pediocin PA-1-like bacteriocin among LAB present in their close vicinity by means of conjugation. The dissemination of pediocin PA-1-like bacteriocin under in situ conditions is noteworthy, and such bacteriocin producers can be useful in the fermentation of dairy products and construction of novel cultures.     

Differentiation between cold shock proteins and cold acclimation proteins in a mesophilic gram-positive bacterium, Enterococcus faecalis JH2-2.

Transfer of Enterococcus faecalis to a cold temperature (8 degrees C for 4 to 30 h) led to increased expression of 11 cold shock proteins (CSPs). Furthermore, this mesophilic prokaryote synthesized 10 cold acclimation proteins, five of them distinct from CSPs, during continuous growth (4 days) at the same temperature (8 degrees C).     

Physiological response of Enterococcus faecalis JH2-2 to cold shock: growth at low temperatures and freezing/thawing challenge

Growth at low positive temperatures and induced phenotypic resistance to extreme cold temperature (freezing/thawing cycles) of Enterococcus faecalis were investigated. The effect of low temperatures on the specific growth rates was studied; use of Arrhenius profile and Ratkovsky 'square-root' model allowed determination of the 'temperature characteristic' (μ≅ 13 800 cal mol^-1), the critical temperature (T_crit≅ 17.9C) and the notional minimum growth temperature (T_o≅ 3.6C). Preincubation of Ent. faecalis cells at low temperatures (8–16C) during periods corresponding to their generation time resulted in an increased ability of the bacterial cells to withstand short periods of freezing/thawing (-20C/+37C) challenge. Moreover, the increase of the incubation period at low positive temperature led to a higher degree of adaptation.     

Physiological and Structural Differences Between Enterococcus faecalis JH2-2 and Mutant Strains Resistant to (P)-Divercin RV41

The aim of this study was to show the differences that could exist at the physiological and structural levels between Enterococcus faecalis JH2-2 (wild type) and three mutant strains resistant to divercin RV41. These mutant strains were recently isolated and characterized for their intermediate resistance to recombinant DvnRV41; a subclass IIa bacteriocin produced by Escherichia coli. These mutant strains were named 35A1 (altered in gene coding phosphoesterase activity), 35H1 (altered in gene coding σ⁵⁴ factor) and 36H4 (altered in gene coding glycerophosphodiesterase). The growth and resistance of each strain were tested against lysozyme. The inhibitory substance did not show any cross-resistance but exhibited an additive effect ascribed to the combined action of lysozyme and (P)-DvnRV41. The use of Fourier transform infrared spectroscopy (FT-IR) allowed to unravelling differences at the structural levels between the aforementioned strains. Thus, mutants 35H1 and 36H4 showed clear differences from mutant 35A1 and wild-type strain. These differences were located, mainly in the fatty acid region and in the polysaccharide composition. This study contributes to understanding more the resistance/sensitivity of Ent. faecalis to (P)-DvnRV41, a subclass IIa bacteriocin.     

Oxalate-degrading Enterococcus faecalis

An oxalate-degrading Enterococcus faecalis was isolated from human stools under anaerobic conditions. The bacteria required a poor nutritional environment and repeated subculturing to maintain their oxalate-degrading ability. The E. faecalis produced 3 proteins (65, 48, and 40 kDa) that were not produced by non-oxalate-degrading E. faecalis as examined by SDS-PAGE. Antibodies against oxalyl-coenzyme A decarboxylase (65 kDa) and formyl-coenzyme A transferase (48 kDa) obtained from Oxalobacter formigenes (an oxalate-degrading anaerobic bacterium in the human intestine) reacted with 2 of the proteins (65 and 48 kDa) from the E. faecalis as examined by Western blottings. This is the first report on the isolation of oxalate-degrading facultative anaerobic bacteria from humans.     

Catabolite repression in Enterococcus faecalis

Metabolism of citrate, pyruvate and sugars by Enterococcus faecalis E-239 and JH2-2 and an isogenic, catabolite derepressed mutant of JH2-2, strain CL4, was investigated. The growth rates of E. faecalis E-239 on citrate and pyruvate were 0.58 and 0.63 h(-1), respectively, indicating that both acids were used as energy sources. Fructose and glucose prevented the metabolism of citrate until all the glucose or fructose had been metabolised. Diauxie growth was not observed but growth on glucose and fructose was much faster than on citrate. In contrast, citrate was co-metabolized with galactose or sucrose and pyruvate with glucose. When glucose was added to cells growing on citrate, glucose metabolism began immediately but inhibition of citrate utilisation did not begin for approximately 1.5 h. Growth rates of E. faecalis JH2-2 and its isogenic, catabolite derepressed mutant, strain CL4, on citrate, were 0.41 and 0.36 h(-1), respectively. The catabolite derepressed mutant was able to co-metabolise citrate and glucose at all concentrations of glucose tested (3-25 mM), while its parent, could only metabolise citrate once all the glucose had been consumed. In strains JH2-2 and E-239, the growth rate on citrate decreased as the glucose concentration increased and, in 25 mM glucose, consumption of citrate was inhibited for several hours after glucose had been consumed. These results indicate that catabolite repression by glucose and fructose occurs in enterococci.     

Genetic diversity among Enterococcus faecalis

Enterococcus faecalis, a ubiquitous member of mammalian gastrointestinal flora, is a leading cause of nosocomial infections and a growing public health concern. The enterococci responsible for these infections are often resistant to multiple antibiotics and have become notorious for their ability to acquire and disseminate antibiotic resistances. In the current study, we examined genetic relationships among 106 strains of E. faecalis isolated over the past 100 years, including strains identified for their diversity and used historically for serotyping, strains that have been adapted for laboratory use, and isolates from previously described E. faecalis infection outbreaks. This collection also includes isolates first characterized as having novel plasmids, virulence traits, antibiotic resistances, and pathogenicity island (PAI) components. We evaluated variation in factors contributing to pathogenicity, including toxin production, antibiotic resistance, polymorphism in the capsule (cps) operon, pathogenicity island (PAI) gene content, and other accessory factors. This information was correlated with multi-locus sequence typing (MLST) data, which was used to define genetic lineages. Our findings show that virulence and antibiotic resistance traits can be found within many diverse lineages of E. faecalis. However, lineages have emerged that have caused infection outbreaks globally, in which several new antibiotic resistances have entered the species, and in which virulence traits have converged. Comparing genomic hybridization profiles, using a microarray, of strains identified by MLST as spanning the diversity of the species, allowed us to identify the core E. faecalis genome as consisting of an estimated 2057 unique genes.     

LuxS-Dependent AI-2 Regulates Versatile Functions in Enterococcus faecalis V583

Bacteria utilize a quorum sensing (QS) system to coordinate gene expression by monitoring the concentration of molecules known as autoinducers (AI). In the present study, we confirmed the presence of a LuxS/AI-2 dependent QS system in vancomycin-resistant Enterococcus faecalis V583. Then, the cellular targets controlled by AI-2 were identified by comparative proteomics analysis in order to elucidate the possible role of AI-2 in E. faecalis. Results demonstrated 15 proteins that are differentially expressed upon the addition of AI-2, including proteins involved in metabolism, translation, energy production and/or conversion, and cell wall biogenesis. Glyceraldehyde-3-phosphate dehydrogenase and malate dehydrogenase associated with carbohydrate metabolism and energy production were up-regulated upon inducing by AI-2. In addition, externally added AI-2 could down-regulate acetyl-coenzyme A carboxylase and ornithine carbamoyltransferase, two key enzyme involved in metabolism. All these data suggest that AI-2 signaling may play a role in the regulation of a number of important metabolic properties of E. faecali. We further investigated the role of AI-2 in biofilm formation by E. faecalis, showing the addition of AI-2 to E. faecalis V583 cultures resulted in increased biofilm formation. Our results provide important clues to the role of a LuxS/AI-2 dependent QS system in vancomycin-resistant E. faecalis.     

Neuroinfections due to Enterococcus faecalis in children

Enterococcal meningitis is a rare complication of neurosurgical procedure or high technology treatment of children and occurs mainly imunocompromised neonates with very low birth weight, severe prematurity and complicates sometime ventriculoperitoneal shunt insertion or perinatal trauma. E. faecalis caused 10 nosocomial meningitis and all strains were susceptible to vancomycin and chloramphenicol, and in our database 90% also to gentamicin and ampicillin. Mortality in our group of 10 children was 20% what is insignificantly higher than overall mortality in the whole cohort of meningitis within last 15 years in our database (15.1%). Early empiric therapy should include also ampicillin or vancomycin, if enterococcal etiology is suspected.     

Characterization of monolaurin resistance in Enterococcus faecalis

There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 microg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.     

Characterization of Monolaurin Resistance in Enterococcus faecalis

There is increasing concern regarding the presence of vancomycin-resistant enterococci in domestically farmed animals, which may act as reservoirs and vehicles of transmission for drug-resistant enterococci to humans, resulting in serious infections. In order to assess the potential for the use of monolaurin as a food preservative, it is important to understand both its target and potential mechanisms of resistance. A Tn917 mutant library of Enterococcus faecalis AR01/DGVS was screened for resistance (MIC, >100 μg/ml) to monolaurin. Three mutants were identified as resistant to monolaurin and were designated DGRM2, DGRM5, and DGRM12. The gene interrupted in all three mutants was identified as traB, which encodes an E. faecalis pheromone shutdown protein and whose complementation in trans restored monolaurin sensitivity in all three mutants. DGRM2 was selected for further characterization. E. faecalis DGRM2 showed increased resistance to gentamicin and chloramphenicol (inhibitors of protein synthesis), while no difference in the MIC was observed with the cell wall-active antibiotics penicillin and vancomycin. E. faecalis AR01/DGVS and DGRM2 were shown to have similar rates (30% cell lysis after 4 h) of cell autolytic activity when activated by monolaurin. Differences in cell surface hydrophobicity were observed between the wild type and the mutant, with the cell surface of the parent strain being significantly more hydrophobic. Analysis of the cell wall structure of DGRM2 by transmission electron microscopy revealed an increase in the apparent cell wall thickness and contraction of its cytoplasm. Taken together, these results suggest that the increased resistance of DGRM2 was due to a change in cell surface hydrophobicity, consequently limiting the diffusion of monolaurin to a potential target in the cytoplasmic membrane and/or cytoplasm of E. faecalis.     

Horizontal transfer of tet(M) and erm(B) resistance plasmids from food strains of Lactobacillus plantarum to Enterococcus faecalis JH2-2 in the gastrointestinal tract of gnotobiotic rats

Two wild-type strains of Lactobacillus plantarum previously isolated from fermented dry sausages were analysed for their ability to transfer antibiotic resistance plasmids in the gastrointestinal tract. For this purpose, we used gnotobiotic rats as an in vivo model. Rats were initially inoculated with the recipient Enterococcus faecalis JH2-2 at a concentration of 10(10) CFU mL(-1). After a week, either of the two donors L. plantarum DG 522 (harbouring a tet(M)-containing plasmid of c. 40 kb) or L. plantarum DG 507 [harbouring a tet(M)-containing plasmid of c. 10 kb and an erm(B)-containing plasmid of c. 8.5 kb] was introduced at concentrations in the range of 10(8)-10(10) CFU mL(-1). Two days after donor introduction, the first transconjugants (TCs) were detected in faecal samples. The detected numbers of tet(M)-TCs were comparable for the two donors. In both cases, this number increased to c. 5 x 10(2) CFU g(-1) faeces towards the end of the experiment. For erm(B)-TCs, the number was significantly higher and increased to c. 10(3) CFU g(-1) faeces. To our knowledge, this is the first study showing in vivo transfer of wild-type antibiotic resistance plasmids from L. plantarum to E. faecalis.     

Enterococcus faecalis Grows on Ascorbic Acid

We show that Enterococcus faecalis can utilize ascorbate for fermentative growth. In chemically defined media, growth yield was limited by the supply of amino acids, and the cells showed a much higher demand for amino acids than when they were grown on glucose.     

Esp-independent biofilm formation by Enterococcus faecalis

Enterococcus faecalis is a gram-positive opportunistic pathogen known to form biofilms in vitro. In addition, this organism is often isolated from biofilms on the surfaces of various indwelling medical devices. However, the molecular mechanisms regulating biofilm formation in these clinical isolates are largely unknown. Recent work has suggested that a specific cell surface protein (Esp) of E. faecalis is critical for biofilm formation by this organism. However, in the same study, esp-deficient strains of E. faecalis were found to be capable of biofilm formation. To test the hypothesis that Esp is dispensable for biofilm formation by E. faecalis, we used microtiter plate assays and a chemostat-based biofilm fermentor assay to examine biofilm formation by genetically well-defined, non-Esp-expressing strains. Our results demonstrate that in vitro biofilm formation occurs, not only in the absence of esp, but also in the absence of the entire pathogenicity island that harbors the esp coding sequence. Using scanning electron microscopy to evaluate biofilms of E. faecalis OG1RF grown in the fermentor system, biofilm development was observed to progress through multiple stages, including attachment of individual cells to the substratum, microcolony formation, and maturation into complex multilayered structures apparently containing water channels. Microtiter plate biofilm analyses indicated that biofilm formation or maintenance was modulated by environmental conditions. Furthermore, our results demonstrate that expression of a secreted metalloprotease, GelE, enhances biofilm formation by E. faecalis. In summary, E. faecalis forms complex biofilms by a process that is sensitive to environmental conditions and does not require the Esp surface protein.     

Characterization of Enterococcus faecalis and Enterococcus faecium from wild flowers

Wild flowers in the South of Spain were screened for Enterococcus faecalis and Enterococcus faecium. Enterococci were frequently associated with prickypear and fieldpoppy flowers. Forty-six isolates, from 8 different flower species, were identified as E. faecalis (28 isolates) or E. faecium (18 isolates) and clustered in well-defined groups by ERIC-PCR fingerprinting. A high incidence of antibiotic resistance was detected among the E. faecalis isolates, especially to quinupristin/dalfopristin (75%), rifampicin (68%) and ciprofloxacin (57%), and to a lesser extent to levofloxacin (35.7%), erythromycin (28.5%), tetracycline (3.5%), chloramphenicol (3.5%) and streptomycin (3.5%). Similar results were observed for E. faecium isolates, except for a higher incidence of resistance to tetracycline (17%) and lower to erythromycin (11%) or quinupristin/dalfopristin (22%). Vancomycin or teicoplanin resistances were not detected. Most isolates (especially E. faecalis) were proteolytic and carried the gelatinase gene gelE. Genes encoding other potential virulence factors (ace, efaA _fs, ccf and cpd) were frequently detected. Cytolysin genes were mainly detected in a few haemolytic E. faecium isolates, three of which also carried the collagen adhesin acm gene. Hyaluronidase gene (hyl _Efm ) was detected in two isolates. Many isolates produced bacteriocins and carried genes for enterocins A, B, and L50 mainly. The similarities found between enterococci from wild flowers and those from animal and food sources raise new questions about the puzzling lifestyle of these commensals and opportunistic pathogens.