Comparative analysis of proteomic adaptations in Enterococcus faecalis and Enterococcus faecium after long term bile acid exposure.

BACKGROUND: All gastrointestinal pathogens, including Enterococcus faecalis and Enterococcus faecium, undergo adaptation processes during colonization and infection. In this study, we investigated by data-independent acquisition mass spectrometry (DIA-MS) two crucial adaptations of these two Enterococcus species at the proteome level. Firstly, we examined the adjustments to cope with bile acid concentrations at 0.05% that the pathogens encounter during a potential gallbladder infection. Therefore, we chose the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) as well as the secondary bile acid deoxycholic acid (DCA), as these are the most prominent bile acids. Secondly, we investigated the adaptations from an aerobic to a microaerophilic environment, as encountered after oral-fecal infection, in the absence and presence of deoxycholic acid (DCA). RESULTS: Our findings showed similarities, but also species-specific variations in the response to the different bile acids. Both Enterococcus species showed an IC50 in the range of 0.01- 0.023% for DCA and CDCA in growth experiments and both species were resistant towards 0.05% CA. DCA and CDCA had a strong effect on down-expression of proteins involved in translation, transcription and replication in E. faecalis (424 down-expressed proteins with DCA, 376 down-expressed proteins with CDCA) and in E. faecium (362 down-expressed proteins with DCA, 391 down-expressed proteins with CDCA). Proteins commonly significantly altered in their expression in all bile acid treated samples were identified for both species and represent a "general bile acid response". Among these, various subunits of a V-type ATPase, different ABC-transporters, multi-drug transporters and proteins related to cell wall biogenesis were up-expressed in both species and thus seem to play an essential role in bile acid resistance. Most of the differentially expressed proteins were also identified when E. faecalis was incubated with low levels of DCA at microaerophilic conditions instead of aerobic conditions, indicating that adaptations to bile acids and to a microaerophilic atmosphere can occur simultaneously. CONCLUSIONS: Overall, these findings provide a detailed insight into the proteomic stress response of two Enterococcus species and help to understand the resistance potential and the stress-coping mechanisms of these important gastrointestinal bacteria.

C-terminal deletion of RelA protein is suggested as a possible cause of infective endocarditis recurrence with Enterococcus faecium.

Infective endocarditis (IE) caused by Enterococcus spp. represents the third most common cause of IE, with high rates of relapse compared with other bacteria. Interestingly, late relapses (>6 months) have only been described in Enterococcus faecalis, but here we describe the first reported IE relapse with Enterococcus faecium more than a year (17 months) after the initial endocarditis episode. Firstly, by multi locus sequence typing (MLST), we demonstrated that both isolates (EF646 and EF641) belong to the same sequence type (ST117). Considering that EF641 was able to overcome starvation and antibiotic treatment conditions surviving for a long period of time, we performed bioinformatic analysis in identifying potential genes involved in virulence and stringent response. Our results showed a 13-nucleotide duplication (positions 1638-1650) in the gene relA, resulting in a premature stop codon, with a loss of 167 amino acids from the C-terminal domains of the RelA enzyme. RelA mediates the stringent response in bacteria, modulating levels of the alarmone guanosine tetraphosphate (ppGpp). The relA mutant (EF641) was associated with lower growth capacity, the presence of small colony variants, and higher capacity to produce biofilms (compared with the strain EF646), but without differences in antimicrobial susceptibility patterns according to standard procedures during planktonic growth. Instead, EF641 demonstrated tolerance to high doses of teicoplanin when growing in a biofilm. We conclude that all these events would be closely related to the long-term survival of the E. faecium and the late relapse of the IE. These data represent the first clinical evidence of mutations in the stringent response (relA gene) related with E. faecium IE relapse.

Study of an Enterococcus faecium strain isolated from an artisanal Mexican cheese, whole-genome sequencing, comparative genomics, and bacteriocin expression.

Enterococci are ubiquitous microorganisms in almost all environments, from the soil we step on to the food we eat. They are frequently found in naturally fermented foods, contributing to ripening through protein, lipid, and sugar metabolism. On the other hand, these organisms are also leading the current antibiotic resistance crisis. In this study, we performed whole-genome sequencing and comparative genomics of an Enterococcus faecium strain isolated from an artisanal Mexican Cotija cheese, namely QD-2. We found clear genomic differences between commensal and pathogenic strains, particularly in their carbohydrate metabolic pathways, resistance to vancomycin and other antibiotics, bacteriocin production, and bacteriophage and CRISPR content. Furthermore, a bacteriocin transcription analysis performed by RT-qPCR revealed that, at the end of the log phase, besides enterocins A and X, two putative bacteriocins not reported previously are also transcribed as a bicistronic operon in E. faecium QD-2, and are expressed 1.5 times higher than enterocin A when cultured in MRS broth.

Effects of Enterococcus faecium (R8a) on nonspecific immune gene expression, immunity and intestinal flora of giant tiger shrimp (Penaeus monodon).

In this study, Penaeus monodon were gave basic feed supplemented with three levels of Enterococcus faecium. Then, the expression of non-specific immunity-related genes, and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (AKP), phenol oxidase (PO) were evaluated. Meanwhile, the disease resistance test and intestinal flora determination were conducted. The results showed that the MDA levels of 2% and 5% E. faecium groups were significantly lower than that of the control group (P < 0.05). While the SOD and T-AOC and ACP and AKP of experimental groups were significantly higher (P < 0.05), the PO of experimental groups were significantly lower than that of the control group (P < 0.05). In addition, the expressions of immunity-related genes (tlr22, dorsal, lysozyme, crustin, imd, and relish) in the 2% and 5% E. faecalis groups were significantly greater than those in the control group (P < 0.05). After P. monodon was challenged with Vibrio parahaemolyticus for 7 days, the average cumulative mortality of P. monodon in the 2% and 5% groups were significantly lower than that in the 0% group (P < 0.05). With the increase of feeding time, the number of effective OTUs in each group showed a downward trend. At the 14th d, Proteobacteria, Bacteroidetes and Firmicutes, the dominant flora in the intestinal tract of P. monodon. In summary, supplied with E. faecium could increase the expression of non-specific immunity-related genes, enhance the immune capacity of P. monodon.

Flow cytometric detection of vancomycin-resistant Enterococcus faecium in urine using fluorescently labelled enterocin K1.

A urinary tract infection (UTI) occurs when bacteria enter and multiply in the urinary system. The infection is most often caused by enteric bacteria that normally live in the gut, which include Enterococcus faecium. Without antibiotic treatment, UTIs can progress to life-threatening septic shock. Early diagnosis and identification of the pathogen will reduce antibiotic use and improve patient outcomes. In this work, we develop and optimize a cost-effective and rapid (< 40 min) method for detecting E. faecium in urine. The method uses a fluorescently labelled bacteriocin enterocin K1 (FITC-EntK1) that binds specifically to E. faecium and is then detected using a conventional flow cytometer. Using this detection assay, urine containing E. faecium was identified by an increase in the fluorescent signals by 25-73-fold (median fluorescence intensity) compared to control samples containing Escherichia coli or Staphylococcus aureus. The method presented in this work is a proof of concept showing the potential of bacteriocins to act as specific probes for the detection of specific bacteria, such as pathogens, in biological samples.

Simple purification and characterization of soluble and homogenous ABC-F translation factors from Enterococcus faecium.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments.

Structural and functional features of a broad-spectrum prophage-encoded enzybiotic from Enterococcus faecium.

Multidrug-resistant (MDR) bacteria have become a growing threat to public health. The gram-positive Enterococcus faecium is classified by WHO as a high-priority pathogen among the global priority list of antibiotic-resistant bacteria. Peptidoglycan-degrading enzymes (PDEs), also known as enzybiotics, are useful bactericidal agents in the fight against resistant bacteria. In this work, a genome-based screening approach of the genome of E. faecium allowed the identification of a putative PDE gene with predictive amidase activity (EfAmi1; EC 3.5.1.28) in a prophage-integrated sequence. EfAmi1 is composed by two domains: a N-terminal Zn2+-dependent N-acetylmuramoyl-L-alanine amidase-2 (NALAA-2) domain and a C-terminal domain with unknown structure and function. The full-length gene of EfAmi1 was cloned and expressed as a 6xHis-tagged protein in E. coli. EfAmi1 was produced as a soluble protein, purified, and its lytic and antimicrobial activities were investigated using turbidity reduction and Kirby-Bauer disk-diffusion assays against clinically isolated bacterial pathogens. The crystal structure of the N-terminal amidase-2 domain was determined using X-ray crystallography at 1.97 Å resolution. It adopts a globular fold with several α-helices surrounding a central five-stranded ß-sheet. Sequence comparison revealed a cluster of conserved amino acids that defines a putative binding site for a buried zinc ion. The results of the present study suggest that EfAmi1 displays high lytic and antimicrobial activity and may represent a promising new antimicrobial in the post-antibiotic era.

The LiaFSR-LiaX System Mediates Resistance of Enterococcus faecium to Peptide Antibiotics and to Aureocin A53- and Enterocin L50-Like Bacteriocins.

Multidrug-resistant Enterococcus faecium strains are currently a leading cause of difficult-to-treat nosocomial infections. The emerging resistance of enterococci to last-resort antibiotics, such as daptomycin, prompts a search for alternative antimicrobials. Aureocin A53- and enterocin L50-like bacteriocins are potent antimicrobial agents that form daptomycin-like cationic complexes and have a similar cell envelope-targeting mechanism of action, suggesting their potential as next-generation antibiotics. However, to ensure their safe use, the mechanisms of resistance to these bacteriocins and cross-resistance to antibiotics need to be well understood. Here, we investigated the genetic basis of E. faecium's resistance to aureocin A53- and enterocin L50-like bacteriocins and compared it with that to antibiotics. First, we selected spontaneous mutants resistant to the bacteriocin BHT-B and identified adaptive mutations in the liaFSR-liaX genes encoding the LiaFSR stress response regulatory system and the daptomycin-sensing protein LiaX, respectively. We then demonstrated that a gain-of-function mutation in liaR increases the expression of liaFSR, liaXYZ, cell wall remodeling-associated genes, and hypothetical genes involved in protection against various antimicrobials. Finally, we showed that adaptive mutations or overexpression of liaSR or liaR alone results in cross-resistance to other aureocin A53- and enterocin L50-like bacteriocins, as well as antibiotics targeting specific components of the cell envelope (daptomycin, ramoplanin, gramicidin) or ribosomes (kanamycin and gentamicin). Based on the obtained results, we concluded that activation of the LiaFSR-mediated stress response confers resistance to peptide antibiotics and bacteriocins via a cascade of reactions, eventually leading to cell envelope remodeling. IMPORTANCE Pathogenic enterococci carry virulence factors and a considerable resistome, which makes them one of the most serious and steadily increasing causes of hospital epidemiological risks. Accordingly, Enterococcus faecium is classified into a top-priority ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of six highly virulent and multidrug-resistant (MDR) bacterial pathogens for which novel antimicrobial agents need to be developed urgently. Alternative measures, such as the use of bacteriocins, separately or in combination with other antimicrobial agents (e.g., antibiotics), could be a potential solution, especially since several international health agencies recommend and support the development of such interventions. Nevertheless, in order to exploit their efficacy, more basic research on the mechanisms of cell killing and the development of resistance to bacteriocins is needed. The present study fills some of the knowledge gaps regarding the genetic basis of the development of resistance to potent antienterococcal bacteriocins, pointing out the common and divergent features regarding the cross-resistance to antibiotics.

Molecular dynamics simulation of pyruvate kinase to investigate improved thermostability of artificially selected strain in Enterococcus faecium.

BACKGROUND: Enterococcus faecium (E. faecium) is a member of symbiotic lactic acid bacteria in gastrointestinal tract and it was successfully used to treat diarrhea cases in humans. For a lactobacteria to survive during the pasteurization process, resistance of proteins to denaturation at high temperatures is crucial. Pyruvate kinase (PYK) is one of the proteins possessing such property. It plays a major role during glycolysis by producing pyruvate and adenosine triphosphate (ATP). OBJECTIVE: To assess the acquired thermostability of PYK of ALE strain using in silico methods. METHODS: First, we predicted and assessed tertiary structures of our proteins using SWISS-MODEL homology modelling server. Second, we then applied molecular dynamics (MD) simulation to simulate and assess multiple properties of molecules. Therefore, we implemented comparative MD to evaluate thermostability of PYK of recently developed high temperature resistant strain of E. faecium using Adaptive Laboratory Evolution (ALE) method. After 20ns of simulation at different temperatures, we observed that ALE enhanced strain demonstrated slightly better stability at 300, 340 and 350 K compared to that of the wild type (WT) strain. RESULTS: We collected the results of MD simulation at four temperature points: 300, 340, 350 and 400 K. Our results showed that the protein demonstrated increased stability at 340 and 350 K. CONCLUSION: Results of these study suggest that PYK of ALE enhanced strain of E. faecium demonstrates overall better stability at elevated temperatures compared to that of WT strain.

Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

Microbiota, Phagocytic Activity, Biochemical Parameters and Parasite Control in Horses with Application of Autochthonous, Bacteriocin-Producing, Probiotic Strain Enterococcus faecium EF 412.

The beneficial influence of bacteriocin-producing, probiotic, mostly non-autochthonous bacteria has already been reported in various animals. However, their use in horses provides limited information, and results with autochthonous bacteria have not been reported. Therefore, the main objective of this model study was to test the effect of autochthonous, bacteriocin-producing faecal strain Enterococcus faecium EF 412 application in horses. One gram of freeze-dried EF 412 strain (109 CFU/mL for 21 days) was applied to horses in a small feed ball. Clinically healthy horses (12), Slovak warm-blood breed of various ages (5-13 years), were involved in a 35-day-long experiment, also functioning as control for themselves. They were stabled in separate boxes (university property), fed twice a day (hay, whole oats or grazed) with water access ad libitum. Sampling was performed at the start of the experiment, i.e. at days 0/1, 21 (3 weeks of EF 412 application) and at day 35 (2 weeks of EF 412 cessation). EF 412 colonized GIT of horses was 3.54 ± 0.75 CFU/g (log 10) at day 21. The eggs of the nematode Strongylus spp. were not found in horses after EF 412 application, and Eimeria spp. oocysts were similarly not found. The other microbiota were not reduced as evaluated by the use of standard method. Using next-generation sequencing, at phylum level, phyla Bacteroidetes and Firmicutes dominated and at family level, they were Bacteroidales BS11 and S24-7 gut goups and Lentisphaerae. In horses, the increasing tendency in phagocytic activity was noted after EF 412 application. Biochemical parameters were in the physiological range. Total protein value was significantly decreased at day 21 compared with day 0/1 as well as with day 35 (P < 0.05). Cholesterol and triglycerides were influenced (decreased) at day 21 compared with day 0/1 and day 35. Neither nematode eggs Strongylus spp. nor Eimeria spp. oocysts were found in faeces after EF 412 application. Autochthonous, faecal strain E. faecium EF 412 showed promising application potential.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Rapid Proteomic Characterization of Bacteriocin-Producing Enterococcus faecium Strains from Foodstuffs.

Enterococcus belongs to a group of microorganisms known as lactic acid bacteria (LAB), which constitute a broad heterogeneous group of generally food-grade microorganisms historically used in food preservation. Enterococci live as commensals of the gastrointestinal tract of warm-blooded animals, although they also are present in food of animal origin (milk, cheese, fermented sausages), vegetables, and plant materials because of their ability to survive heat treatments and adverse environmental conditions. The biotechnological traits of enterococci can be applied in the food industry; however, the emergence of enterococci as a cause of nosocomial infections makes their food status uncertain. Recent advances in high-throughput sequencing allow the subtyping of bacterial pathogens, but it cannot reflect the temporal dynamics and functional activities of microbiomes or bacterial isolates. Moreover, genetic analysis is based on sequence homologies, inferring functions from databases. Here, we used an end-to-end proteomic workflow to rapidly characterize two bacteriocin-producing Enterococcus faecium (Efm) strains. The proteome analysis was performed with liquid chromatography coupled to a trapped ion mobility spectrometry-time-of-flight mass spectrometry instrument (TimsTOF) for high-throughput and high-resolution characterization of bacterial proteins. Thus, we identified almost half of the proteins predicted in the bacterial genomes (>1100 unique proteins per isolate), including quantifying proteins conferring resistance to antibiotics, heavy metals, virulence factors, and bacteriocins. The obtained proteomes were annotated according to function, resulting in 22 complete KEGG metabolic pathway modules for both strains. The workflow used here successfully characterized these bacterial isolates and showed great promise for determining and optimizing the bioengineering and biotechnology properties of other LAB strains in the food industry.

In vitro virulence activity of Pseudomonas aeruginosa, enhanced by either Acinetobacter baumannii or Enterococcus faecium through the polymicrobial interactions.

Microbes within an infection impact neighbors' pathogenicity. This study aimed to address in vitro virulence activity of Pseudomonas aeruginosa under the binary interaction with Acinetobacter baumannii or Enterococcus faecium, co-isolated from two chronic wound infections. The biofilm formation of Pseudomonas was enhanced 1.5- and 1.4-fold when it was simultaneously cultured with Acinetobacter and Enterococcus, respectively. Pseudomonas motility was increased by 1.9- and 1.5-fold (swimming), 3.6- and 1.9-fold (swarming), and 1.5- and 1.5-fold (twitching) in the dual cultures with Acinetobacter and Enterococcus, respectively. The synergistic hemolysis activity of Pseudomonas was observed with the heat-killed Acinetobacter and Enterococcus cells. The minimum inhibitory concentration of ciprofloxacin against Pseudomonas was increased from (µg mL-1) 25 to 400 in the individual and mixed cultures, respectively. The pyocyanin production by Pseudomonas in the single and mixed cultures with Acinetobacter and Enterococcus was (µg/mL) 1.8, 2.3, and 2.9, respectively. The expression of lasI, rhlI, and pqsR genes was up-regulated by 1.0-, 1.9-, and 16.3-fold, and 4.9-, 1.0-, and 9.3-fold when Pseudomonas was incubated with Acinetobacter and Enterococcus, respectively. Considering the entire community instead of a single pathogen may lead to a more effective therapeutic design for persistent infections caused by Pseudomonas.

The extracellular domain of site-2-metalloprotease RseP is important for sensitivity to bacteriocin EntK1.

Enterocin K1 (EntK1), a bacteriocin that is highly potent against vancomycin-resistant enterococci, depends on binding to an intramembrane protease of the site-2 protease family, RseP, for its antimicrobial activity. RseP is highly conserved in both EntK1-sensitive and EntK1-insensitive bacteria, and the molecular mechanisms underlying the interaction between RseP and EntK1 and bacteriocin sensitivity are unknown. Here, we describe a mutational study of RseP from EntK1-sensitive Enterococcus faecium to identify regions of RseP involved in bacteriocin binding and activity. Mutational effects were assessed by studying EntK1 sensitivity and binding with strains of naturally EntK1-insensitive Lactiplantibacillus plantarum-expressing various RseP variants. We determined that site-directed mutations in conserved sequence motifs related to catalysis and substrate binding, and even deletion of two such motifs known to be involved in substrate binding, did not abolish bacteriocin sensitivity, with one exception. A mutation of a highly conserved asparagine, Asn359, in the extended so-called LDG motif abolished both binding of and killing by EntK1. By constructing various hybrids of the RseP proteins from sensitive E. faecium and insensitive L. plantarum, we showed that the extracellular PDZ domain is the key determinant of EntK1 sensitivity. Taken together, these data may provide valuable insight for guided construction of novel bacteriocins and may contribute to establishing RseP as an antibacterial target.

Exopolysaccharide of Enterococcus faecium L15 promotes the osteogenic differentiation of human dental pulp stem cells via p38 MAPK pathway.

BACKGROUND: Bone has important functions in the body. Several researchers have reported that the polysaccharides and lipopolysaccharide derived from microbes can promote osteogenic differentiation of stem cells. Enterococcus faecium, a lactic acid bacterium (LAB), produces several bioactive metabolites and has been widely applied in the food and nutraceutical industries. The exopolysaccharide (EPS) from LAB has also been extensively examined for its postbiotic effects and for its in vivo and in vitro functionalities. However, studies on promoting bone differentiation using polysaccharides from LAB are lacking. Therefore, the purpose of this study was to investigate the effect of E. faecium L15 extract and EPS on osteogenic differentiation of human dental pulp stem cells (hDPSCs) and to identify the underlying mechanisms. METHODS: hDPSCs were obtained from dental pulp tissue, and L15 extract and EPS were isolated from L15. Gene and protein expression of the osteogenic differentiation markers were analyzed with qPCR and western blotting and the possible signaling pathways were also investigated using western blotting. Osteogenic differentiation potential was examined by alkaline phosphatase (ALP) staining and alizarin red s (ARS) staining. In addition, osteogenic differentiation potential of L15 EPS was explored in ex vivo culture of neonate murine calvaria. RESULTS: The calcium deposition and ALP activity were enhanced by addition of L15 extract or EPS. The expression levels of RUNX2, ALP, and COL1A1 mRNA and the protein expression levels of RUNX2, ALP, and BMP4 were increased in hDPSCs treated with the L15 extract or EPS. The L15 EPS treatment enhanced phosphorylation of the p38 mitogen-activated protein kinase (MAPK). The L15 EPS-induced increases in RUNX2, ALP, and BMP4 expression were suppressed by the p38 MAPK inhibitor SB203580. The promoting effect of L15 EPS on osteogenic differentiation was not only seen in hDPSCs, but also in osteoblast precursors. ALP activity and the expression of RUNX2, ALP, and COL1A1 increased in the L15 EPS-treated osteoblast precursors. In addition, L15 EPS increased bone thickness of neonate murine calvaria in ex vivo culture. CONCLUSIONS: The stimulatory effect of L15 extract and EPS on osteogenic differentiation occurred through the p38 MAPK pathway, and L15 EPS enhanced new bone formation in neonate murine calvaria. These data suggest that L15 EPS has therapeutic potential applicable to bone regeneration.

Purification and biochemical characterization of a new thermostable laccase from Enterococcus faecium A2 by a three-phase partitioning method and investigation of its decolorization potential.

Three-phase partitioning (TPP) is a simple, fast, cost-effective, and highly efficient process that can be used in the purification of laccases. In this study, microorganisms with laccase activity were isolated from water samples collected from the Agri-Diyadin hot spring. The isolate with the highest laccase activity was found to be the A2 strain. As a result of molecular (16S rRNA sequence) and conventional (morphological, biochemical, and physiological) analyses, it was determined that the A2 isolate was 99% similar to Enterococcus faecium (Genbank number: MH424896). The laccase was purified to 4.9-fold with 110% recovery using the TPP. The molecular mass of the enzyme was found by SDS-PAGE to be 50.11 kDa. Optimum pH 6.0 and optimum temperature for laccase were determined as 80 °C. The laccase exhibited pH stability over a wide range (pH 3.0-9.0) and a high thermostability, retaining over 90% of its activity after 1 h of incubation at 20-90 °C. The laccase exhibited high thermostability, with a heat inactivation half-life of approximately 24 h at 80 °C. The enzyme remained highly stable in the presence of surfactants and increased its activity in the presence of organic solvents, Cr2+, Cu2+, and Ag+ metal ions. The Km, Vmax, kcat, and kcat/Km values of laccase for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate were 0.68 mM, 5.29 µmol mL-1 min-1, 110.2 s-1, and 162.1 s-1 mM-1, respectively.

Proteomics reveal the protective effects of chlorogenic acid on Enterococcus faecium Q233 in a simulated pro-oxidant colonic environment.

Certain phytochemicals have been found to promote the beneficial effects of probiotic bacteria although the molecular mechanisms of such interactions are poorly understood. The objective of the present study was to evaluate the impact of the exposure to 0.5 mM chlorogenic acid (CA) on the redox status and proteome of Enterococcus faecium isolated from cheese and challenged with 2.5 mM hydrogen peroxide (H2O2). The bacterium was incubated in anaerobic conditions for 48 h at 37 °C. CA exposure led to a more intense oxidative stress and accretion of bacterial protein carbonyls than those induced by H2O2. The oxidative damage to bacterial proteins was even more severe in the bacterium treated with both CA and H2O2, yet, such combination led to a strengthening of the antioxidant defenses, namely, a catalase-like activity. The proteomic study indicated that H2O2 caused a decrease in energy supply and the bacterium responded by reinforcing the membrane and wall structures and counteracting the redox and pH imbalance. CA stimulated the accretion of proteins related to translation and transcription regulators, and hydrolases. This phytochemical was able to counteract certain proteomic changes induced by H2O2 (i.e. increase of ATP binding cassete (ABC) transporter complex) and cause the increase of Rex, a redox-sensitive protein implicated in controlling metabolism and responses to oxidative stress. Although this protection should be confirmed under in vivo conditions, such effects point to benefits in animals or humans affected by disorders in which oxidative stress plays a major role.

Psychobiotic Potential of Gamma-Aminobutyric Acid-Producing Marine Enterococcus faecium SH9 from Marine Shrimp.

Psychobiotics are a novel class of probiotics with potential to confer mental wellness via production of neuroactive compounds such as gamma-aminobutyric acid (GABA). The demand for new biological sources of GABA has increased steadily. Therefore, the current study reports the isolation of 17 presumptive lactic acid bacteria (LAB) from marine samples and their screening for GABA synthesis from monosodium glutamate (MSG) using thin-layer chromatography (TLC). The isolate SH9 was selected as a high GABA producing strain. The GABA content of SH9 cell free supernatant (CFS) was quantitatively determined by high performance liquid chromatography (HPLC) to be 0.97 g/L. SH9 was identified biochemically and molecularly as Enterococcus faecium (identity 99%). Moreover, SH9 demonstrated promising probiotic potentials; it gave no signs of hemolysis and could survive at low pH values and high bile salt concentrations. It also exhibited antimicrobial activity against highly pathogenic strains and the ability to grow at 6.5% NaCl. In addition, SH9 CFS showed anti-inflammatory and antioxidant properties. The glutamate decarboxylase (GAD) gene was detected in SH9 by using specific primers. Product of 540 bp was obtained, sequenced, and analyzed (accession number: MW713382). The inferred amino acid sequence was 99.3% identical to Lactobacillus plantarum M-6 gadB gene. The findings of this study suggest that the marine isolate E. faecium SH9 could be used as a novel psychobiotics in the development of GABA rich healthy products.

Purification, Characterization, Mode of Action, and Application of Jileicin, a Novel Antimicrobial from Paenibacillus jilinensis YPG26.

Antimicrobial compounds from the commensal gut microbiota have gained much attention due to their multifunctionality in maintaining good health in the host and killing multidrug-resistant bacteria. Our previous study showed that Paenibacillus jilinensis YPG26 isolated from chicken intestine can antagonize multiple pathogens. Herein, we characterized a bacteriocin-like inhibitory substance, jileicin, purified from P. jilinensis YPG26. Mass spectrometry analysis revealed that jileicin was a protein consisting of 211 amino acids, which showed 88.98% identity to the SIMPL domain-containing protein. The jileicin showed a relatively broad-spectrum antibacterial ability, especially against enterococci. Additionally, the jileicin exhibited good stability after various treatments, no detectable resistance, no significant cytotoxicity, and very low levels of hemolytic activity. The mode of action against Enterococcus faecium demonstrated that jileicin could destroy cell membrane integrity, increase cell membrane permeability, and eventually lead to cell death. Furthermore, jileicin was efficient in controlling the growth of E. faecium in milk. In conclusion, jileicin, as a newly identified antibacterial agent, is expected to be a promising candidate for application in the food, pharmaceutical, and biomedical industries.