Survey on phenotypic resistance in Enterococcus faecalis: comparison between the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells.

BACKGROUND: Phenotypic resistance is considered as a serious therapeutic challenge for which a definitive remedy has not been discovered yet. Biofilm and persister cell formation are two well-studied phenotypic resistance phenomena, leading to the recalcitrance and relapse of different types of chronic infections. The presence of persister cells in biofilm structures seems to be one of the main factors contributing to the relapse of infections and treatment failure. Given the dormant and inert nature of persister cells, they can be easy targets for the immune system factors. Biofilm formation can be a survival strategy for the defenseless persister cells. Thus, this study was aimed to evaluate the expression of biofilm-associated genes in Enterococcus faecalis persister and non-persister cells. METHODS: Vancomycin susceptibility and biofilm formation ability were investigated among 95 E. faecalis clinical isolates using microtiter broth dilution and microtiter plate assays, respectively. PCR was used to determine the presence of biofilm-related genes (gelE, esp, and agg) among the vancomycin-susceptible, biofilm producer E. faecalis isolates (91 isolates). Minimum bactericidal concentration for biofilms (MBCB) were determined for vancomycin using the MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biofilm-related genes was compared between the persister and non-persister isolates of E. faecalis using real-time qPCR. RESULTS: E. faecalis isolates showed a high level of susceptibility (95.8%) to vancomycin (MIC < 1 µg/mL). The gelE, esp, and agg genes were found in 91 (100%), 72 (79.12), and 74 (81.32) of the isolates, respectively. All the E. faecalis isolates were tolerant to vancomycin in the biofilm condition, showing a MBCB of > 2500 µg/mL. Based on the enzymatic lysis assay, only 3 isolates, out of the 91, had the ability to form persister cells. The expression of biofilm-associated genes was higher among the persister compared to non-persister E. faecalis isolates. CONCLUSIONS: Biofilm-associated persister cells indicated a high vancomycin tolerance compared to non-persister cells. Moreover, persister isolates showed a higher tendency for biofilm formation and a higher expression level of the biofilm-associated genes, compared to non-persister isolates.

MICy: a Novel Flow Cytometric Method for Rapid Determination of Minimal Inhibitory Concentration.

Early initiated adequate antibiotic treatment is essential in intensive care. Shortening the length of antibiotic susceptibility testing (AST) can accelerate clinical decision-making. Our objective was to develop a simple flow cytometry (FC)-based AST that produces reliable results within a few hours. We developed a FC-based AST protocol (MICy) and tested it on six different bacteria strains (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis) in Mueller-Hinton and Luria-Bertani broth. We monitored the bacterial growth by FC to define the optimal time of AST. All bacteria were tested against 12 antibiotics and the MIC values were compared to microdilution used as reference method. McNemar and Fleiss' kappa inter-observer tests were performed to analyze the bias between the two methods. Susceptibility profiles of the two methods were also compared. We found that FC is able to detect the bacterial growth after 4-h incubation. The point-by-point comparison of MICy and microdilution resulted in exact match above 87% (2642/3024) of all measurements. The MIC values obtained by MICy and microdilution agreed over 80% (173/216) within ±1 dilution range that gives a substantial inter-observer agreement with weighted Fleiss' kappa. By using the EUCAST clinical breakpoints, we defined susceptibility profiles of MICy that were identical to microdilution in more than 92% (197/213) of the decisions. MICy resulted 8.7% major and 3.2% very major discrepancies. MICy is a new, simple FC-based AST method that produces susceptibility profile with low failure rate a workday earlier than the microdilution method. IMPORTANCE MICy is a new, simple and rapid flow cytometry based antibiotic susceptibility testing (AST) method that produces susceptibility profile a workday earlier than the microdilution method or other classical phenotypic AST methods. Shortening the length of AST can accelerate clinical decision-making as targeted antibiotic treatment improves clinical outcomes and reduces mortality, duration of artificial ventilation, and length of stay in intensive care unit. It can also reduce nursing time and costs and the spreading of antibiotic resistance. In this study, we present the workflow and methodology of MICy and compare the results produced by MICy to microdilution step by step.

Ultrasonic agitation reduces the time of calcium hydroxide antimicrobial effect and enhances its penetrability.

OBJECTIVES: The objective of the present work was to evaluate the ultrasonic agitation, time and vehicle (propylene glycol or distilled water) on the antimicrobial potential and penetrability of calcium hydroxide pastes on infected dentin by means of Confocal Laser Scanning Microscopy (CLSM) and microbiological culture (MC). MATERIALS AND METHODS: Dentin specimens were infected with Enterococcus faecalis using a new contamination protocol of 5 days. The specimens were divided into eight groups and dressed with the pastes for 7 or 15 days: G1) calcium hydroxide (CH) + propylene glycol (prop)/7 days (d), G2) CH + prop/7d + ultrasonic agitation (U), G3) CH + distilled water (dw)/7d, G4) CH + dw/7d + U, G5) CH + prop/15d, G6) CH + prop/15d + U, G7) CH + dw/15d, G8) CH + dw/15d + U. The ultrasonic activation was made for 1 min in both directions with a plain point insert. After medications removal, the images obtained by CLSM showed the viable (green) and dead (red) bacteria with Live and Dead dye. By the MC, the dentinal wall debris obtained by burs were collected for colony counts. For the penetration test, the Rodamine B dye was added to the CH pastes and analyzed by CLSM. RESULTS: The 7 and 15-days CH + prop+U pastes performed better antimicrobial efficacy, followed by the CH + dw+U/15d paste. CONCLUSIONS: All pastes demonstrated better penetration and antimicrobial activity against E. faecalis when agitated with ultrasound, even in periods of up to seven days. The propylene glycol vehicle showed better results. CLINICAL RELEVANCE: Agitation of the dressing that remains for less time inside the root canal can optimize the decontamination of endodontic treatment.

Antimicrobial Effect of Calcium Hydroxide Combined with Electrolyzed Superoxidized Solution at Neutral pH on Enterococcus faecalis Growth.

OBJECTIVE: To evaluate the effect of the combination of calcium hydroxide (Ca(OH)2) and a novel electrolyzed superoxidized solution at neutral pH, known as OxOral® on Enterococcus faecalis growth in root canals. METHODS: Sixty human teeth were used, from which root canals were infected and randomly divided into the following treatment groups: saline solution, saline solution plus Ca(OH)2, OxOral®, and OxOral® plus Ca(OH)2. RESULTS: A permanent reduction in bacterial growth was observed at days 1, 6, 12, and 18 after OxOral® plus Ca(OH)2 treatment from 4.4 ± 0.074 log10 CFU/mL to 0.0 ± 0.001 log10 CFU/mL. In addition, alkaline conditions maintenance was observed from application time (pH = 12.2 ± 0.033) to 18 d posttreatment (pH = 12.6 ± 0.083). CONCLUSION: The combination of OxOral® and Ca(OH)2 provides an alkaline pH and inhibits E. faecalis growth into the root canals. Our study opens the possibility for further research on the use of OxOral® in endodontic therapy.

Structural Transformation and Creativity Induced by Biological Agents during Fermentation of Edible Nuts from Terminalia catappa.

Terminalia catappa L. (tropical almond) is a nutritious fruit found mainly in the tropics. This study is aimed to establish the naturally biotransformed molecules and identify the probiotic agents facilitating the fermentation. The aqueous extracts from both the unfermented and fermented T. catappa nuts were subjected to gas chromatography/mass spectrometry (GC/MS) analysis. Syringol (6.03%), glutamine (1.71%), methyl laurate (1.79%), methyl palmitate (1.53%), palmitic acid (5.20%), palmitoleic acid (2.80%), and methyl oleate (2.97%) were detected in the unfermented nuts of the T. catappa. Additionally, two of these natural compounds (palmitic acid (4.19%) and palmitoleic acid (1.48%)) survived the fermentation process to emerge in the fermented seeds. The other natural compounds were biotransformed into 2,3-butanediol (1.81%), butyric acid (16.20%), propane-1,3-diol (19.66%), neoheptanol (2.89%), 2-piperidinone (6.63%), palmitoleic acid (1.18%), formamide, n-(p-hydroxyphenethyl)- (2.80%), and cis-vaccenic acid (1.69%) that newly emerged in the fermented seeds. The phytochemical compounds are likely carbon sources for the organisms facilitating the biotransformed molecules and product production. Four (4) potential probiotic bacteria strains, namely, Probt B1a, Probt B2a, Probt B4a, and Probt B4b, were isolated from the fermented nut. Enterococcus faecum, and Enterococcus faecalis were the organisms identified as driving the fermentation of the seeds. All strains were gram-positive, catalase-negative, and non-hemolytic, which suggests their harmless nature. N-(p-hydroxyphenethyl)-) was associated with fermentation for the first time, and neoheptanol was discovered as the main alcoholic molecule formed during the fermentation of the seeds. This fermentation is a handy tool for bio-transforming compounds in raw food sources into compounds with nutritious and therapeutic potentials.

Selection and Identification of Novel Antibacterial Agents against Planktonic Growth and Biofilm Formation of Enterococcus faecalis.

YycFG, one of the two-component systems involved in the regulation of biofilm formation, has attracted increasing interest as a potential target of antibacterial and antibiofilm agents. YycG inhibitors for Staphylococcus aureus and Staphylococcus epidermidis have been developed, but Enterococcus faecalis remains underexplored. Herein, we selected and identified novel candidate molecules against E. faecalis targeting histidine kinase YycG using high-throughput virtual screening; six molecules (compound-16, -30, -42, -46, -59, and -62) with low cytotoxicity toward mammalian cells were verified as potential YycG inhibitors through an autophosphorylation test and binding kinetics. Compound-16 inhibited planktonic cells of E. faecalis, including the vancomycin- or linezolid-resistant strains. In contrast, compound-62 did not affect planktonic growth but significantly inhibited biofilm formation in static and dynamic conditions. Compound-62 combined with ampicillin could synergistically eradicate the biofilm-embedded viable bacteria. The study demonstrates that YycG inhibitors may be valuable approaches for the development of novel antimicrobial agents for difficult-to-treat bacterial infections.

Repurposing of the Tamoxifen Metabolites to Treat Methicillin-Resistant Staphylococcus epidermidis and Vancomycin-Resistant Enterococcus faecalis Infections.

Repurposing drugs provides a new approach to the fight against multidrug-resistant (MDR) bacteria. We have reported that three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), presented bactericidal activity against Acinetobacter baumannii and Escherichia coli. Here, we aimed to analyze the activity of a mixture of the three tamoxifen metabolites against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus species. MRSE (n = 17) and Enterococcus species (Enterococcus faecalis n = 8 and Enterococcus faecium n = 10) strains were used. MIC of the mixture of DTAM, HTAM, and ENDX and that of vancomycin were determined by microdilution assay. The bactericidal activity of the three metabolites together and of vancomycin against MRSE (SE385 and SE742) and vancomycin-resistant E. faecalis (EVR1 and EVR2) strains was determined by time-kill curve assays. Finally, changes in membrane permeability of SE742 and EVR1 strains were analyzed using fluorescence assays. MIC90 of tamoxifen metabolites was 1 mg/liter for MRSE strains and 2 mg/liter for E. faecalis and E. faecium strains. In the time-killing assays, tamoxifen metabolites mixture showed bactericidal activity at 4× MIC for MRSE (SE385 and SE742) and at 2× MIC and 4× MIC for E. faecalis (EVR1 and EVR2) strains, respectively. SE385 and EVR2 strains treated with the tamoxifen metabolites mixture presented higher membrane permeabilization. Altogether, these results showed that tamoxifen metabolites presented antibacterial activity against MRSE and vancomycin-resistant E. faecalis, suggesting that tamoxifen metabolites might increase the arsenal of drug treatments against these bacterial pathogens. IMPORTANCE The development of new antimicrobial therapeutic strategies requires immediate attention to avoid the tens of millions of deaths predicted to occur by 2050 as a result of MDR bacterial infections. In this study, we assessed the antibacterial activity of three major tamoxifen metabolites, N-desmethyltamoxifen (DTAM), 4-hydroxytamoxifen (HTAM), and endoxifen (ENDX), against methicillin-resistant Staphylococcus epidermidis (MRSE) and Enterococcus spp. (E. faecalis and E. faecium). We found that the tamoxifen metabolites have antibacterial activity against MRSE, E. faecalis, and E. faecium strains by presenting MIC90 between 1 and 2 mg/liter and bactericidal activity over 24 h. In addition, this antibacterial activity is paralleled by an increased membrane permeability of these strains. Our results showed that tamoxifen metabolites might be potentially used as a therapeutic alternative when treating MRSE and E. faecalis strains in an animal model of infection.

Evaluation of antimicrobial and anticancer activities of three peptides identified from the skin secretion of Hylarana latouchii.

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs.

Synergism versus Additivity: Defining the Interactions between Common Disinfectants.

Many of the most common disinfectant and sanitizer products are formulations of multiple antimicrobial compounds. Products claiming to contain synergistic formulations are common, although there is often little supporting evidence. The antimicrobial interactions of all pairwise combinations of common disinfectants (benzalkonium chloride, didecyldimethylammonium chloride, polyhexamethylene biguanide, chlorocresol, and bronopol) were classified via checkerboard assay and validated by time-kill analyses. Combinations were tested against Acinetobacter baumannii NCTC 12156, Enterococcus faecalis NCTC 13379, Klebsiella pneumoniae NCTC 13443, and Staphylococcus aureus NCTC 13143. Synergistic interactions were identified only for the combinations of chlorocresol with benzalkonium chloride and chlorocresol with polyhexamethylene biguanide. Synergism was not ubiquitously demonstrated against all species tested and was on the borderline of the synergism threshold. These data demonstrate that synergism between disinfectants is uncommon and circumstantial. Most of the antimicrobial interactions tested were characterized as additive. We suggest that this is due to the broad, nonspecific mechanisms associated with disinfectants not providing an opportunity for the combined activities of these compounds to exceed the sum of their parts. IMPORTANCE The scarcity of observed synergistic interactions suggests that in the case of many disinfectant-based products, combined mechanisms of interaction may be being misinterpreted. We emphasize the need to correctly differentiate between additivity and synergism in antimicrobial formulations, as inappropriate classification may lead to unnecessary issues in the event of regulatory changes. Furthermore, we question the need to focus on synergism and disregard additivity when considering combinations of disinfectants, as the benefits that synergistic interactions provide are not necessarily relevant to the application of the final product.

c-di-AMP Is Essential for the Virulence of Enterococcus faecalis.

Second messenger nucleotides are produced by bacteria in response to environmental stimuli and play a major role in the regulation of processes associated with bacterial fitness, including but not limited to osmoregulation, envelope homeostasis, central metabolism, and biofilm formation. In this study, we uncovered the biological significance of c-di-AMP in the opportunistic pathogen Enterococcus faecalis by isolating and characterizing strains lacking genes responsible for c-di-AMP synthesis (cdaA) and degradation (dhhP and gdpP). Using complementary approaches, we demonstrated that either complete loss of c-di-AMP (ΔcdaA strain) or c-di-AMP accumulation (ΔdhhP, ΔgdpP, and ΔdhhP ΔgdpP strains) drastically impaired general cell fitness and virulence of E. faecalis. In particular, the ΔcdaA strain was highly sensitive to envelope-targeting antibiotics, was unable to multiply and quickly lost viability in human serum or urine ex vivo, and was virtually avirulent in an invertebrate (Galleria mellonella) and in two catheter-associated mouse infection models that recapitulate key aspects of enterococcal infections in humans. In addition to evidence linking these phenotypes to altered activity of metabolite and peptide transporters and inability to maintain osmobalance, we found that the attenuated virulence of the ΔcdaA strain also could be attributed to a defect in Ebp pilus production and activity that severely impaired biofilm formation under both in vitro and in vivo conditions. Collectively, these results demonstrate that c-di-AMP signaling is essential for E. faecalis pathogenesis and a desirable target for drug development.

Densely Functionalized 2-Methylideneazetidines: Evaluation as Antibacterials.

Twenty-two novel, variously substituted nitroazetidines were designed as both sulfonamide and urethane vinylogs possibly endowed with antimicrobial activity. The compounds under study were obtained following a general procedure recently developed, starting from 4-nitropentadienoates deriving from a common ß-nitrothiophenic precursor. While being devoid of any activity against fungi and Gram-negative bacteria, most of the title compounds performed as potent antibacterial agents on Gram-positive bacteria (E. faecalis and three strains of S. aureus), with the most potent congener being the 1-(4-chlorobenzyl)-3-nitro-4-(p-tolyl)azetidine 22, which displayed potency close to that of norfloxacin, the reference antibiotic (minimum inhibitory concentration values 4 and 1-2 µg/mL, respectively). Since 22 combines a relatively efficient activity against Gram-positive bacteria and a cytotoxicity on eucharyotic cells only at 4-times higher concentrations (inhibiting concentration on 50% of the cultured eukaryotic cells: 36 ± 10 µM, MIC: 8.6 µM), it may be considered as a promising hit compound for the development of a new series of antibacterials selectively active on Gram-positive pathogens. The relatively concise synthetic route described herein, based on widely available starting materials, could feed further structure-activity relationship studies, thus allowing for the fine investigation and optimization of the toxico-pharmacological profile.

Mechanism of the Immunomodulatory Effect of the Combination of Live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus on Immunocompromised Rats.

Immunodeficiency is a very common condition in suboptimal health status and during the development or treatment of many diseases. Recently, probiotics have become an important means for immune regulation. The present study aimed to investigate the mechanism of the immunomodulatory effect of a combination of live Bifidobacterium, Lactobacillus, Enterococcus, and Bacillus (CBLEB), which is a drug used by approximately 10 million patients every year, on cyclophosphamide-immunosuppressed rats. Cyclophosphamide (40 mg/kg) was intraperitoneally injected to induce immunosuppression in a rat model on days 1, 2, 3, and 10. Starting from day 4, the rats were continuously gavaged with CBLEB solution for 15 days. The samples were collected to determine routine blood test parameters, liver and kidney functions, serum cytokine levels, gut microbiota, fecal and serum metabolomes, transcriptomes, and histopathological features. The results indicated that CBLEB treatment reduced cyclophosphamide-induced death, weight loss, and damage to the gut, liver, spleen, and lungs and eliminated a cyclophosphamide-induced increase in the mean hemoglobin content and GGT, M-CSF, and MIP-3α levels and a decrease in the red blood cell distribution width and total protein and creatinine levels in the blood. Additionally, CBLEB corrected cyclophosphamide-induced dysbiosis of the gut microbiota and eliminated all cyclophosphamide-induced alterations at the phylum level in rat feces, including the enrichment in Proteobacteria, Fusobacteriota, and Actinobacteriota and depletion of Spirochaetota and Cyanobacteria. Furthermore, CBLEB treatment alleviated cyclophosphamide-induced alterations in the whole fecal metabolome profile, including enrichment in 1-heptadecanol, succinic acid, hexadecane-1,2-diol, nonadecanoic acid, and pentadecanoic acid and depletion of benzenepropanoic acid and hexane. CBLEB treatment also alleviated cyclophosphamide-induced enrichment in serum D-lyxose and depletion of serum succinic acid, D-galactose, L-5-oxoproline, L-alanine, and malic acid. The results of transcriptome analysis indicated that the mechanism of the effect of CBLEB was related to the induction of recovery of cyclophosphamide-altered carbohydrate metabolism and signal transduction. In conclusion, the present study provides an experimental basis and comprehensive analysis of application of CBLEB for the treatment of immunodeficiency.

Prodigiosin inhibits bacterial growth and virulence factors as a potential physiological response to interspecies competition.

Prodigiosin, a red linear tripyrrole pigment, has long been recognised for its antimicrobial property. However, the physiological contribution of prodigiosin to the survival of its producing hosts still remains undefined. Hence, the aim of this study was to investigate the biological role of prodigiosin from Serratia marcescens, particularly in microbial competition through its antimicrobial activity, towards the growth and secreted virulence factors of four clinical pathogenic bacteria (methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa) as well as Staphylococcus aureus and Escherichia coli. Prodigiosin was first extracted from S. marcescens and its purity confirmed by absorption spectrum, high performance liquid chromatography (HPLC) and liquid chromatography-tandem mass spectrophotometry (LC-MS/MS). The extracted prodigiosin was antagonistic towards all the tested bacteria. A disc-diffusion assay showed that prodigiosin is more selective towards Gram-positive bacteria and inhibited the growth of MRSA, S. aureus and E. faecalis and Gram-negative E. coli. A minimum inhibitory concentration of 10 µg/µL of prodigiosin was required to inhibit the growth of S. aureus, E. coli and E. faecalis whereas > 10 µg/µL was required to inhibit MRSA growth. We further assessed the effect of prodigiosin towards bacterial virulence factors such as haemolysin and production of protease as well as on biofilm formation. Prodigiosin did not inhibit haemolysis activity of clinically associated bacteria but was able to reduce protease activity for MRSA, E. coli and E. faecalis as well as decrease E. faecalis, Salmonella Typhimurium and E. coli biofilm formation. Results of this study show that in addition to its role in inhibiting bacterial growth, prodigiosin also inhibits the bacterial virulence factor protease production and biofilm formation, two strategies employed by bacteria in response to microbial competition. As clinical pathogens were more resistant to prodigiosin, we propose that prodigiosin is physiologically important for S. marcescens to compete against other bacteria in its natural soil and surface water environments.

Antimicrobial Photodynamic Inactivation Affects the Antibiotic Susceptibility of Enterococcus spp. Clinical Isolates in Biofilm and Planktonic Cultures.

Enterococcus faecium and Enterococcus faecalis are opportunistic pathogens that can cause a vast variety of nosocomial infections. Moreover, E. faecium belongs to the group of ESKAPE microbes, which are the main cause of hospital-acquired infections and are especially difficult to treat because of their resistance to many antibiotics. Antimicrobial photodynamic inactivation (aPDI) represents an alternative to overcome multidrug resistance problems. This process requires the simultaneous presence of oxygen, visible light, and photosensitizing compounds. In this work, aPDI was used to resensitize Enterococcus spp. isolates to antibiotics. Antibiotic susceptibility testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations was combined with synergy testing methods recommended by the American Society for Microbiology. Two clinical isolates, E. faecalis and E. faecium, were treated with a combination of aPDI utilizing rose bengal (RB) or fullerene (FL) derivative as photosensitizers, antimicrobial blue light (aBL), and 10 recommended antibiotics. aPDI appeared to significantly impact the survival rate of both isolates, while aBL had no significant effect. The synergy testing results differed between strains and utilized methods. Synergy was observed for RB aPDI in combination with gentamycin, ciprofloxacin and daptomycin against E. faecalis. For E. faecium, synergy was observed between RB aPDI and gentamycin or ciprofloxacin, while for RB aPDI with vancomycin or daptomycin, antagonism was observed. A combination of FL aPDI gives a synergistic effect against E. faecalis only with imipenem. Postantibiotic effect tests for E. faecium demonstrated that this isolate exposed to aPDI in combination with gentamycin, streptomycin, tigecycline, doxycycline, or daptomycin exhibits delayed growth in comparison to untreated bacteria. The results of synergy testing confirmed the effectiveness of aPDI in resensitization of the bacteria to antibiotics, which presents great potential in the treatment of infections caused by multidrug-resistant strains.

Peptide/ß-Peptoid Hybrids with Activity against Vancomycin-Resistant Enterococci: Influence of Hydrophobicity and Structural Features on Antibacterial and Hemolytic Properties.

Infections with enterococci are challenging to treat due to intrinsic resistance to several antibiotics. Especially vancomycin-resistant Enterococcus faecium and Enterococcus faecalis are of considerable concern with a limited number of efficacious therapeutics available. From an initial screening of 20 peptidomimetics, 11 stable peptide/ß-peptoid hybrids were found to have antibacterial activity against eight E. faecium and E. faecalis isolates. Microbiological characterization comprised determination of minimal inhibitory concentrations (MICs), probing of synergy with antibiotics in a checkerboard assay, time-kill studies, as well as assessment of membrane integrity. E. faecium isolates proved more susceptible than E. faecalis isolates, and no differences in susceptibility between the vancomycin-resistant (VRE) and -susceptible E. faecium isolates were observed. A test of three peptidomimetics (Ac-[hArg-ßNsce]6-NH2, Ac-[hArg-ßNsce-Lys-ßNspe]3-NH2 and Oct-[Lys-ßNspe]6-NH2) in combination with conventional antibiotics (vancomycin, gentamicin, ciprofloxacin, linezolid, rifampicin or azithromycin) revealed no synergy. The same three potent analogues were found to have a bactericidal effect with a membrane-disruptive mode of action. Peptidomimetics Ac-[hArg-ßNsce-Lys-ßNspe]3-NH2 and Oct-[Lys-ßNspe]6-NH2 with low MIC values (in the ranges 2-8 µg/mL and 4-16 µg/mL against E. faecium and E. faecalis, respectively) and displaying weak cytotoxic properties (i.e., <10% hemolysis at a ~100-fold higher concentration than their MICs; IC50 values of 73 and 41 µg/mL, respectively, against HepG2 cells) were identified as promising starting points for further optimization studies.

Antibacterial composite coatings of MgB2 powders embedded in PVP matrix.

Three commercial powders of MgB2 were tested in vitro by MTS and LDH cytotoxicity tests on the HS27 dermal cell line. Depending on powders, the toxicity concentrations were established in the range of 8.3-33.2 µg/ml. The powder with the lowest toxicity limit was embedded into polyvinylpyrrolidone (PVP), a biocompatible and biodegradable polymer, for two different concentrations. The self-replenishing MgB2-PVP composite materials were coated on substrate materials (plastic foil of the reservoir and silicon tubes) composing a commercial urinary catheter. The influence of the PVP-reference and MgB2-PVP novel coatings on the bacterial growth of Staphylococcus aureus ATCC 25923, Enterococcus faecium DMS 13590, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, in planktonic and biofilm state was assessed in vitro at 6, 24, and 48 h of incubation time. The MgB2-PVP coatings are efficient both against planktonic microbes and microbial biofilms. Results open promising applications for the use of MgB2 in the design of anti-infective strategies for different biomedical devices and systems.

Synthesis, Characterization, and Antimicrobial Activity of CoO Nanoparticles from a Co (II) Complex Derived from Polyvinyl Alcohol and Aminobenzoic Acid Derivative.

Cobalt oxide nanoparticles (CoO NPs) were synthesized by the calcination method from the Co (II) complex which has the formula [Co(PVA)(P-ABA)(H2O)3], PVA = polyvinyl alcohol, and P-ABA = para-aminobenzoic acid. The calcination temperature was 550°C, and the products were characterized by element analysis, thermal analyses (TGA and DTA), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), UV-Vis spectra, and scanning electron microscopy (SEM) techniques. The kinetic and thermodynamic parameters (∆H ∗ , ∆G ∗ , and ∆S ∗ ) for the cobalt (II) complex are calculated. The charges been carried by the atoms cause dipole moment 10.53 and 3.84 debye and total energy 11.04 × 102 and 24.80 × 102 k Cal mol-1 for the Co (II) complex and cobalt oxide, respectively. X-ray diffraction confirmed that the resulting oxide was pure single-crystalline CoO nanoparticles. Scanning electron microscopy indicating that the crystallite size of cobalt oxide nanocrystals was in the range of 36-54 nm. Finally, the antimicrobial activity of cobalt oxide nanoparticles was evaluated using four bacterial strains and one fungal strain. Two strains of Gram-positive cocci (Staphylococcus aureus and Enterococcus faecalis), two strains of Gram-negative bacilli (Escherichia coli and Pseudomonas aeruginosa), and one strain of yeast such as fungi (Candida albicans) were used in this study.

Oxygen radical antioxidant capacity (ORAC) and antibacterial properties of Melicope glabra bark extracts and isolated compounds.

Melicope glabra (Blume) T. G. Hartley from the Rutaceae family is one of the richest sources of plant secondary metabolites, including coumarins and flavanoids. This study investigates the free radical scavenging and antibacterial activities of M. glabra and its isolated compounds. M. glabra ethyl acetate and methanol extracts were prepared using the cold maceration technique. The isolation of compounds was performed with column chromatography. The free radical scavenging activity of the extracts and isolated compounds were evaluated based on their oxygen radical absorbance capacity (ORAC) activities. The extracts and compounds were also subjected to antibacterial evaluation using bio-autographic and minimal inhibitory concentration (MIC) techniques against two oral pathogens, Enterococcus faecalis and Streptococcus mutans. Isolation of phytoconstituents from ethyl acetate extract successfully yielded quercetin 3, 5, 3'-trimethyl ether (1) and kumatakenin (2), while the isolation of the methanol extract resulted in scoparone (3), 6, 7, 8-trimethoxycoumarin (4), marmesin (5), glabranin (6), umbelliferone (7), scopoletin (8), and sesamin (9). The study is the first to isolate compound (1) from Rutaceae plants, and also the first to report the isolation of compounds (2-5) from M. glabra. The ORAC evaluation showed that the methanol extract is stronger than the ethyl acetate extract, while umbelliferone (7) exhibited the highest ORAC value of 24 965 µmolTE/g followed by glabranin (6), sesamin (9) and scopoletin (8). Ethyl acetate extract showed stronger antibacterial activity towards E. faecalis and S. mutans than the methanol extract with MIC values of 4166.7 ± 1443.4 µg/ml and 8303.3 ± 360.8 µg/ml respectively. Ethyl acetate extract inhibited E. faecalis growth, as shown by the lowest optical density value of 0.046 at a concentration of 5.0 mg/mL with a percentage inhibition of 95%. Among the isolated compounds tested, umbelliferone (7) and sesamin (9) exhibited promising antibacterial activity against S. mutans with both exhibiting MIC values of 208.3 ± 90.6 µg/ml. Findings from this study suggests M. glabra as a natural source of potent antioxidant and antibacterial agents.

Comparison of Enterococcus faecalis Biofilm Removal Efficiency among Bacteriophage PBEF129, Its Endolysin, and Cefotaxime.

Enterococcus faecalis is a Gram-positive pathogen which colonizes human intestinal surfaces, forming biofilms, and demonstrates a high resistance to many antibiotics. Especially, antibiotics are less effective for eradicating biofilms and better alternatives are needed. In this study, we have isolated and characterized a bacteriophage, PBEF129, infecting E. faecalis. PBEF129 infected a variety of strains of E. faecalis, including those exhibiting antibiotic resistance. Its genome is a linear double-stranded DNA, 144,230 base pairs in length. Its GC content is 35.9%. The closest genomic DNA sequence was found in Enterococcus phage vB_EfaM_Ef2.3, with a sequence identity of 99.06% over 95% query coverage. Furthermore, 75 open reading frames (ORFs) were functionally annotated and five tRNA-encoding genes were found. ORF 6 was annotated as a phage endolysin having an L-acetylmuramoyl-l-alanine amidase activity. We purified the enzyme as a recombinant protein and confirmed its enzymatic activity. The endolysin's host range was observed to be wider than its parent phage PBEF129. When applied to bacterial biofilm on the surface of in vitro cultured human intestinal cells, it demonstrated a removal efficacy of the same degree as cefotaxime, but much lower than its parent bacteriophage.

Two ABC transport systems carry out peptide uptake in Enterococcus faecalis: Their roles in growth and in uptake of sex pheromones.

Enterococcal pheromone-inducible plasmids encode a predicted OppA-family secreted lipoprotein. In the case of plasmid pCF10, the protein is PrgZ, which enhances the mating response to cCF10 pheromone. OppA proteins generally function with associated OppBCDF ABC transporters to import peptides. In this study, we analyzed the potential interactions of PrgZ with two host-encoded Opp transporters using two pheromone-inducible fluorescent reporter constructs. Based on our results, we propose renaming these loci opp1 (OG1RF_10634-10639) and opp2 (OG1RF_12366-12370). We also examined the ability of the Opp1 and Opp2 systems to mediate import in the absence of PrgZ. Cells expressing PrgZ were able to import pheromone if either opp1 or opp2 was functional, but not if both opp loci were disrupted. In the absence of PrgZ, pheromone import was dependent on a functional opp2 system, including opp2A. Comparative structural analysis of the peptide-binding pockets of PrgZ, Opp1A, Opp2A, and the related Lactococcus lactis OppA protein, suggested that the robust pheromone-binding ability of PrgZ relates to a nearly optimal fit of the hydrophobic peptide, whereas binding ability of Opp2A likely results from a more open, promiscuous peptide-binding pocket similar to L. lactis OppA.