Faecalis Populations Research Articles

Comparative survival study of Bacillus coagulans and Enterococcus faecium microencapsulated in chitosan-alginate nanoparticles in simulated gastrointestinal condition

Probiotics have poor gastrointestinal delivery because they lose their viability during intestinal passage. Microencapsulation has a significant effect on the survival of probiotic bacteria. This study aimed to synthesize chitosan-alginate nanoparticles and encapsulate Bacillus coagulans NBRC-12583 and Enterococcus faecium MGFR1 in alginate (Alg), chitosan (Cht) and inulin (Inu) by micro- and nano-encapsulation to investigate the viability of encapsulated strains in simulated gastrointestinal condition. The survival of free and encapsulated bacteria was studied for 120 min. The population of B. coagulans encapsulated in Cht-Alg only decreased by 0.86 log CFU, while that of E. faecium encapsulated in Cht-Alg nanoparticles only decreased by 0.27 log CFU. Treated with simulated intestinal fluid, B. coagulans and E. faecium populations decreased by 1.03 and 0.17 log CFU, respectively. E. faecium microencapsulated in Cht-Alg and Inu had a maximum encapsulation efficiency of 96.78%. E. faecium encapsulated in Cht-Alg nanoparticles and Inu was more viable than other encapsulated bacteria. Scanning electron microscopy was done to investigate the microcapsules' surface morphology, structure, internal cross-sectional view, and the zeta potential for the surface charge of Cht-Alg nanoparticles. The viability of the probiotic bacteria was enhanced significantly by microencapsulation and nanomaterial-based encapsulation within the chitosan-alginate and inulin matrix.

Bacteriophage and antibiotic combination therapy for recurrent Enterococcus faecium bacteremia.

Phage therapy is an emerging therapeutic approach for treating bacterial infections that do not respond to traditional antibiotics. The addition of phage therapy to systemic antibiotics to treat a patient with recurrent E. faecium infections that were non-responsive to antibiotics alone resulted in fewer hospitalizations and improved the patient's quality of life. Combination phage and antibiotic therapy reduced E. faecium and VRE abundance in the patient's stool. Eventually, an anti-phage antibody response emerged that was able to neutralize phage activity, which may have limited clinical efficacy. This study demonstrates the potential of phages as an additional option in the antimicrobial toolbox for treating invasive enterococcal infections and highlights the need for further investigation to ensure phage therapy can be deployed for maximum clinical benefit.

Enterococcus faecalis Strains with Compromised CRISPR-Cas Defense Emerge under Antibiotic Selection for a CRISPR-Targeted Plasmid.

Enterococcus faecalis is a Gram-positive bacterium that natively colonizes the human gastrointestinal tract and opportunistically causes life-threatening infections. Multidrug-resistant (MDR) E. faecalis strains have emerged that are replete with mobile genetic elements (MGEs). Non-MDR E. faecalis strains frequently possess CRISPR-Cas systems, which reduce the frequency of MGE acquisition. We demonstrated in previous studies that E. faecalis populations can transiently maintain both a functional CRISPR-Cas system and a CRISPR-Cas target. In this study, we used serial passage and deep sequencing to analyze these populations. In the presence of antibiotic selection for the plasmid, mutants with compromised CRISPR-Cas defense and enhanced ability to acquire a second antibiotic resistance plasmid emerged. Conversely, in the absence of selection, the plasmid was lost from wild-type E. faecalis populations but not E. faecalis populations that lacked the cas9 gene. Our results indicate that E. faecalis CRISPR-Cas can become compromised under antibiotic selection, generating populations with enhanced abilities to undergo horizontal gene transfer. IMPORTANCE Enterococcus faecalis is a leading cause of hospital-acquired infections and disseminator of antibiotic resistance plasmids among Gram-positive bacteria. We have previously shown that E. faecalis strains with an active CRISPR-Cas system can prevent plasmid acquisition and thus limit the transmission of antibiotic resistance determinants. However, CRISPR-Cas is not a perfect barrier. In this study, we observed populations of E. faecalis with transient coexistence of CRISPR-Cas and one of its plasmid targets. Our experimental data demonstrate that antibiotic selection results in compromised E. faecalis CRISPR-Cas function, thereby facilitating the acquisition of additional resistance plasmids by E. faecalis.

Occult Vancomycin-Resistant Enterococcus faecium ST117 Displaying a Highly Mutated vanB2 Operon

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the "classic" occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB2 operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB2 resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB2-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB2-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB2-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.

Adaptation of the gut pathobiont Enterococcus faecalis to deoxycholate and taurocholate bile acids

Enterococcus faecalis is a natural inhabitant of the human gastrointestinal tract. This bacterial species is subdominant in a healthy physiological state of the gut microbiota (eubiosis) in adults, but can become dominant and cause infections when the intestinal homeostasis is disrupted (dysbiosis). The relatively high concentrations of bile acids deoxycholate (DCA) and taurocholate (TCA) hallmark eubiosis and dysbiosis, respectively. This study aimed to better understand how E. faecalis adapts to DCA and TCA. We showed that DCA impairs E. faecalis growth and possibly imposes a continuous adjustment in the expression of many essential genes, including a majority of ribosomal proteins. This may account for slow growth and low levels of E. faecalis in the gut. In contrast, TCA had no detectable growth effect. The evolving transcriptome upon TCA adaptation showed the early activation of an oligopeptide permease system (opp2) followed by the adjustment of amino acid and nucleotide metabolisms. We provide evidence that TCA favors the exploitation of oligopeptide resources to fuel amino acid needs in limiting oligopeptide conditions. Altogether, our data suggest that the combined effects of decreased DCA and increased TCA concentrations can contribute to the rise of E. faecalis population during dysbiosis.

An orally administered drug prevents selection for antibiotic-resistant bacteria in the gut during daptomycin therapy.

Background and objectivesPreviously, we showed proof-of-concept in a mouse model that oral administration of cholestyramine prevented enrichment of daptomycin-resistant Enterococcus faecium in the gastrointestinal (GI) tract during daptomycin therapy. Cholestyramine binds daptomycin in the gut, which removes daptomycin selection pressure and so prevents the enrichment of resistant clones. Here, we investigated two open questions related to this approach: (i) can cholestyramine prevent the enrichment of diverse daptomycin mutations emerging de novo in the gut? and (ii) how does the timing of cholestyramine administration impact its ability to suppress resistance?MethodologyMice with GI E. faecium were treated with daptomycin with or without cholestyramine, and E. faecium was cultured from feces to measure changes in daptomycin susceptibility. A subset of clones was sequenced to investigate the genomic basis of daptomycin resistance.ResultsCholestyramine prevented the enrichment of diverse resistance mutations that emerged de novo in daptomycin-treated mice. Whole-genome sequencing revealed that resistance emerged through multiple genetic pathways, with most candidate resistance mutations observed in the clsA gene. In addition, we observed that cholestyramine was most effective when administration started prior to the first dose of daptomycin. However, beginning cholestyramine after the first daptomycin dose reduced the frequency of resistant E. faecium compared to not using cholestyramine at all.Conclusions and implicationsCholestyramine prevented the enrichment of diverse daptomycin-resistance mutations in intestinal E. faecium populations during daptomycin treatment, and it is a promising tool for managing the transmission of daptomycin-resistant E. faecium.

Fitness cost of vancomycin-resistant Enterococcus faecium plasmids associated with hospital infection outbreaks.

Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds. Widespread Tn1546-carrying plasmids of different families RepA_N (n = 5), Inc18 (n = 4) and/or pHTβ (n = 1), and prototype plasmids RepA_N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison. All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA_N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness. The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains.

Isolation and Characterization of Enterococcus faecalis-Infecting Bacteriophages From Different Cheese Types.

Enterococci are a diverse group of Gram-positive, lactic acid bacteria (LAB). They are found in many environments, including fermented foods, in which they could constitute a health threat since they produce biogenic amines, which consumption can lead to intoxication. Moreover, enterococci has also emerged as an important hospital-acquired pathogens via its acquisition of antimicrobial resistance. Bacteriophages possess features that make them optimal biotechnological weapons for controlling bacterial growth in disease and food spoilage contexts. However, no silver bullet bacteriophage exists that can eliminate all the undesirable bacteria in a complex environment. Rather, a combination of phages with different host ranges would be required which implies the need for large collections of diverse phages. This work reports the isolation of several Enterococcus faecalis-infecting bacteriophages from different types of cheese, along with the range of E. faecalis strains of diverse origin (from dairy to clinical environments) they are able to infect. The isolated phages showed a large diversity regarding the number and origin of strains they infect. Some of these phages were subjected to morphological and genomic characterization which confirmed their diversity and showed they belong to different families and genera. The present findings increase the potential arsenal for the bacteriophage-based biocontrol of harmful E. faecalis populations.

In Vivo Microbial Coevolution Favors Host Protection and Plastic Downregulation of Immunity.

Microbiota can protect their hosts from infection. The short timescales in which microbes can evolve presents the possibility that “protective microbes” can take-over from the immune system of longer-lived hosts in the coevolutionary race against pathogens. Here, we found that coevolution between a protective bacterium (Enterococcus faecalis) and a virulent pathogen (Staphylococcus aureus) within an animal population (Caenorhabditis elegans) resulted in more disease suppression than when the protective bacterium adapted to uninfected hosts. At the same time, more protective E. faecalis populations became costlier to harbor and altered the expression of 134 host genes. Many of these genes appear to be related to the mechanism of protection, reactive oxygen species production. Crucially, more protective E. faecalis populations downregulated a key immune gene, , known to be effective against S. aureus infection. These results suggest that a microbial line of defense is favored by microbial coevolution and may cause hosts to plastically divest of their own immunity.

Antibacterial and antibiofilm activities of thyme oil against foodborne multiple antibiotics-resistant Enterococcus faecalis

The inhibitory and bactericidal activities of thyme oil against the foodborne multiple antibiotics-resistant Enterococcus faecalis biofilm were evaluated in this study. Gas chromatography-mass spectrometry revealed that more than 70% of the composition of thyme oil is thymol. Crystal violet staining assay showed that 128 and 256 μg/mL thyme oil significantly inhibited the biofilm formation of E. faecalis. The cell adherence of E. faecalis, as shown by its swimming and swarming motilities, was reduced by thyme oil. The exopolysaccharide (EPS) quantification assay showed that thyme oil inhibited the EPS synthesis in E. faecalis biofilms. The 3D-view observations through confocal laser scanning and scanning electron microscopy suggested that cell adherence and biofilm thickness were decreased in thyme oil–treated biofilms. Quantitative real-time analyses showed that the transcription of ebp and epa gene clusters, which were related to cell mobility and EPS production, was inhibited by thyme oil. Thus, thyme oil effectively inhibited the biofilm formation of E. faecalis by affecting cell adherence and EPS synthesis. Furthermore, 2,048 and 4,096 μg/mL thyme oil can effectively inactivate E. faecalis population in the mature E. faecalis biofilms by 5.75 and 7.20 log CFU/mL, respectively, after 30 min of treatment. Thus, thyme oil at different concentrations can be used as an effective antibiofilm or germicidal agent to control E. faecalis biofilms.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link.

poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996-2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30-100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

Fluorescent reporter plasmids for single-cell and bulk-level composition assays in E. faecalis.

Fluorescent reporters are an important tool for monitoring dynamics of bacterial populations at the single cell and community level. While there are a large range of reporter constructs available–particularly for common model organisms like E. coli–fewer options exist for other species, including E. faecalis, a gram-positive opportunistic pathogen. To expand the potential toolkit available for E. faecalis, we exchanged the original fluorescent reporter in a previously developed plasmid (pBSU101) with one of eight fluorescent reporters and confirmed that all constructs exhibited detectable fluorescence in single E. faecalis cells and mixed biofilm communities. To identify promising constructs for bulk-level experiments, we then measured the fluorescence spectra from E. faecalis populations in microwell plate (liquid) cultures during different phases of aerobic growth. Cultures showed density- and reporter-specific variations in fluorescent signal, though spectral signatures of all reporters become clear in late-exponential and stationary-phase populations. Based on these results, we identified six pairs of reporters that can be combined with simple spectral unmixing to accurately estimate population composition in 2-strain mixtures at or near stationary phase. This approach offers a simple and scalable method for selection and competition experiments in simple two-species populations under aerobic growth conditions. Finally, we incorporated codon-optimized variants of blue (BFP) and red (RFP) reporters and show that they lead to increased fluorescence in exponentially growing cells. As a whole, the results inform the scope of application of different reporters and identify both single reporters and reporter pairs that are promising for fluorescence-based assays at bulk and single-cell levels in E. faecalis.

Comparative Evaluation of the Efficacy of Novel Root Canal Irrigation Techniques on Reduction of Enterococcus faecalis Count: An In Vitro Study

To compare the effectiveness of three irrigation systems, namely, Endovac system, Max I probe, and Navitip FX, in reduction of Enterococcus faecalis population from the root canal using agar diffusion method. Sixty extracted intact human permanent maxillary anterior teeth were selected for this study. In group I, root canals were irrigated using brush covered 30-gauge NaviTipFX. Ultradent in group II root canals was irrigated using brush covered 30-gauge Max-I-Probe Dentsply. In group III, root canals were irrigated using Endoactivator, Dentsply. In group IV, root canal was irrigated by using the Endovac system Sybronendo. The steps followed in the study include preparation of specimen, contamination of the samples followed by conduction of testing procedures with implementation of appropriate irrigation protocols, and sampling procedures. Data were subjected to statistical analysis to interpret the significant differences among various irrigation systems. One-way analysis of variance, Post hoc Tukey tests were used for statistical analysis in the present study. Among the experimental groups, group IV showed statistically significant difference in reduction of E. faecalis. There were no statistical differences between them in reduction of E. faecalis in group I and group II compared and represented in Tables 1 and 2. All four irrigation delivery systems have been found to be effective in the reduction of E. faecalis. Endovac showed comparable efficacy in reduction of colony-forming units to that of other delivery systems used in the study. The result has to be validated with in vivo studies and clinical trials of larger sample size. Selection of appropriate irrigation system capable of disinfection of canal complexities in apical third with less adverse effects is essential for good clinical success of endodontic treatment.

Genomic, Antimicrobial Resistance, and Public Health Insights into Enterococcus spp. from Australian Chickens.

Due to Australia's management of antimicrobial use in poultry, particularly the discontinued use of avoparcin for nearly 20 years, it is hypothesized that vancomycin-resistant enterococci associated with human disease are not derived from poultry isolates. This study evaluated antimicrobial resistance (AMR) of five enterococcal species isolated from Australian meat chickens, genomic features of Enterococcus faecium and Enterococcus faecalis, and the phylogenetic relationship of the poultry-derived E. faecium with isolates from human sepsis cases. All enterococcal isolates from chicken ceca were subjected to antimicrobial susceptibility testing. E. faecium and E. faecalis underwent whole-genome sequencing. E. faecium was compared at the core genome level to a collection of human isolates (n = 677) obtained from cases of sepsis over a 2-year period spanning 2015 to 2016. Overall, 205 enterococci were isolated consisting of five different species. E. faecium was the most frequently isolated species (37.6%), followed by E. durans (29.7%), E. faecalis (20%), E. hirae (12.2%), and E. gallinarum (0.5%). All isolates were susceptible to vancomycin and gentamicin, while one isolate was linezolid resistant (MIC 16 mg/liter). Core genome analysis of the E. faecium demonstrated two clades consisting predominantly of human or chicken isolates in each clade, with minimal overlap. Principal component analysis for total gene content revealed three clusters comprised of vanA-positive, vanB-positive, and both vanA- and vanB-negative E. faecium populations. The results of this study provide strong evidence that Australian chicken E. faecium isolates are unlikely to be precursor strains to the currently circulating vancomycin-resistant strains being isolated in Australian hospitals.

Investigation of different sodium hypochlorite volumes, concentrations and times of irrigation in endodontic therapy: a systematic review

Investigation of different sodium hypochlorite volumes, concentrations and times of irrigation in endodontic therapy: a systematic review

Evaluation of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing

Salmonella is capable of surviving in a low moisture environment for long periods. Once adapted to the xeric conditions, the thermal resistance of Salmonella is enhanced. Cocoa powder is a low water activity (aw) food (LawF) that is an essential component in a wide variety of desserts and ready-to-eat (RTE) foods and drinks. The aim of this study was to evaluate the desiccation and thermal resistance of Salmonella in cocoa powder, as well as to examine the suitability of Enterococcus faecium NRRL B-2354 as a surrogate for Salmonella during cocoa powder thermal processing. Natural unsweetened cocoa powder was inoculated with a 3-strain Salmonella cocktail or E. faecium and was equilibrated to aw 0.30 and 0.45 at room temperature, then subjected to isothermal treatments at 70–80 °C or 12-month storage at RT (room temperature, 22.0 ± 0.5 °C, aw 0.30). At 70 and 80 °C, D-values of both Salmonella and E. faecium increased with decreasing aw. D-values of Salmonella at aw 0.30 cocoa powder were 46.2 ± 4.7, 20.5 ± 1.7, and 11.5 ± 0.9 min at 70, 75 and 80 °C, respectively. Higher heat resistance of E. faecium in aw 0.30 cocoa powder was observed with D-values of 59.9 ± 5.0, 28.9 ± 1.8, and 16.1 ± 1.4 min at 70, 75, and 80 °C, respectively. However, E. faecium demonstrated less heat resistance than Salmonella when aw was increased to 0.45. D-values for Salmonella at aw 0.45 were 31.6–7.0 min at 70–80 °C compared to 25.8–4.7 min for E. faecium. During 12 months of storage at RT, surviving E. faecium population in aw 0.30 cocoa powder was higher than that of the Salmonella cocktail; the population decreased by 1.39 and 3.75 logs, respectively. These findings indicate that the suitability of E. faecium as a surrogate organism for Salmonella is influenced by aw of cocoa powder. The aw correlates with thermal inactivation rates in both Salmonella and E. faecium, and should be considered as a significant contributor to the thermal resistance of Salmonella in cocoa powder.

Emergence of unusual vanA/vanB2 genotype in a highly mutated vanB2-vancomycin-resistant hospital-associated E. faecium background in Vietnam

Enterococcus faecium has become a globally disseminated nosocomial pathogen mainly because of acquisition and diffusion of virulence factors and multidrug resistance determinants, including glycopeptides, which are some of the last resort antimicrobials used to treat more serious infections common in high-risk patients.In this study we investigated and characterized hospital-associated (HA) E. faecium isolates collected at Hue Central Hospital, Vietnam.Our results highlighted the spread among hospital wards of a surprisingly heterogeneous multidrug-resistant E. faecium population comprising five different CC17-related sequence types (STs), of which 46% VREf carry the vanB gene. Whole genome sequencing of selected E. faecium isolates showed that VREf from different STs carried the same chromosomal integrated Tn1549-like transposon, with a highly mutated vanB2-operon, showing an increased level of vancomycin resistance (VanB phenotype) and able, in one isolate, to confer resistance to teicoplanin (VanA incongruent phenotype).Two unusual vanA/vanB2-type strains were detected within the vanB2-type ST17 population, harbouring a Tn1546-vanA-like transposon in pJEG40-like plasmids. Wg-SNPs-based analysis showed the genetic relatedness of VSEf/VREf of the same STs and indicated lateral exchange of the Tn1549-like element among isolates followed by clonal expansion. Microevolution among ST17 isolates, including the vanA/vanB2-type strains, and inter-wards VREf transmission, were highlighted.The use of teicoplanin is strongly discouraged in the study hospital because of the spreading of Tn1549–vanB2 associated to teicoplanin resistance. A rational use of glycopeptides and effective surveillance measures are required to reduce nosocomial VSEF/VREf spread and to avoid the rise of unusual and misleading VREf genotypes.

Antimicrobial Activity of Phenyllactic Acid Against Enterococcus faecalis and Its Effect on Cell Membrane.

3-Phenyllactic acid (PLA) was reported to have an effective antimicrobial activity. This study evaluated the antimicrobial activity of PLA against foodborne Enterococcus faecalis and its effect on cell membrane. The minimum concentration of PLA to inactivate E. faecalis in brain heart infusion broth was 5 mg/mL. PLA solutions of 5 and 10 mg/mL can inactivate E. faecalis population ≥6 log CFU/mL within 60 and 30 min, respectively. The cell membranes of most E. faecalis cells were damaged after PLA treatment according to the images of scanning electron microscopy and transmission electron microscopy. The differences in the regions of cell membrane protein, fatty acid, and polysaccharide were revealed by Fourier transform infrared spectroscopy, which further indicated cell membrane damages. The cell membrane permeability was increased when the concentration of PLA treatment was increased in the membrane permeability assays. Finally, almost all bacterial cells were damaged after treatment with 10 mg/mL PLA for 30 min, further confirmed by flow cytometry analysis. This study concluded that PLA is effective in inactivating E. faecalis cells through the leakage of intracellular components caused by cell membrane damage.

Novel de novo synthesized phosphate carrier compound ABA-PEG20k-Pi20 suppresses collagenase production in Enterococcus faecalis and prevents colonic anastomotic leak in an experimental model.

Previous work has demonstrated that anastomotic leak can be caused by collagenolytic bacteria such as Enterococcus faecalis via an effect on wound collagen. In humans, E. faecalis is the organism cultured most commonly from a leaking anastomosis, and is not routinely eliminated by standard oral or intravenous antibiotics. Novel strategies are needed to contain the virulence of this pathogen when present on anastomotic tissues. Polyphosphorylated polymer ABA-PEG20k-Pi20 was tested in mice for its ability to prevent anastomotic leak caused by collagenolytic E. faecalis. The study design included a distal colonic resection and anastomosis followed by introduction of E. faecalis to anastomotic tissues via enema. Mice were assigned randomly to receive either ABA-PEG20-Pi20 or its unphosphorylated precursor ABA-PEG20k in their drinking water. The development of anastomotic leak was determined after the animals had been killed. Overnight incubation of two different E. faecalis collagenolytic strains with 2 mmol/l of ABA-PEG20k-Pi20 led to near complete inhibition of collagenase production (from 21 000 to 1000 and from 68 000 to 5000 units; P < 0·001; 6 samples per group) without suppressing bacterial growth. In mice drinking 1 per cent ABA-PEG20k-Pi20, the phosphate concentration in the distal colonic mucosa increased twofold and leak rates decreased from eight of 15 to three of 15 animals (P < 0·001). In mice drinking ABA-PEG20k-Pi20, the percentage of collagenolytic colonies among E. faecalis populations present at anastomotic tissue sites was decreased by 6-4800-fold (P = 0·008; 5 animals). These data indicate that oral intake of ABA-PEG20k-Pi20 may be an effective agent to contain the virulence of E. faecalis and may prevent anastomotic leak caused by this organism. Clinical relevance Progress in understanding the pathogenesis of anastomotic leak continues to point to intestinal bacteria as key causative agents. The presence of pathogens such as Enterococcus faecalis that predominate on anastomotic tissues despite antibiotic use, coupled with their ability to produce collagenase, appears to alter the process of healing that leads to leakage. Further antibiotic administration may seem logical, but carries the unwanted risk of eliminating the normal microbiome, which functions competitively to exclude and suppress the virulence of pathogens such as E. faecalis. Therefore, non-antibiotic strategies that can suppress the production of collagenase by E. faecalis without affecting its growth, or potentially normal beneficial microbiota, may have unique advantages. The findings of this study demonstrate that drinking a phosphate-based polymer can achieve the goal of preventing anastomotic leak by suppressing collagenase production in E. faecalis without affecting its growth.

Transferable vancomycin resistance in clade B commensal-type Enterococcus faecium.

Vancomycin-resistant Enterococcus faecium is a leading cause of MDR hospital infection. Two genetically definable populations of E. faecium have been identified: hospital-adapted MDR isolates (clade A) and vancomycin-susceptible commensal strains (clade B). VanN-type vancomycin resistance was identified in two isolates of E. faecium recovered from blood and faeces of an immunocompromised patient. To understand the genomic context in which VanN occurred in the hospitalized patient, the risk it posed for transmission in the hospital and its origins, it was of interest to determine where these strains placed within the E. faecium population structure. We obtained the genome sequence of the VanN isolates and performed comparative and functional genomics of the chromosome and plasmid content. We show that, in these strains, VanN occurs in a genetic background that clusters with clade B E. faecium, which is highly unusual. We characterized the chromosome and the conjugative plasmid that carries VanN resistance in these strains, pUV24. This plasmid exhibits signatures of in-host selection on the vanN operon regulatory system, which are associated with a constitutive expression of vancomycin resistance. VanN resistance in clade B strains may go undetected by current methods. We report a case of vancomycin resistance in a commensal lineage of E. faecium responsible for an atypical bacteraemia in an immunocompromised patient. A reservoir of transferable glycopeptide resistance in the community could pose a concern for public health.